U.S. patent application number 11/338863 was filed with the patent office on 2007-05-10 for ligand that has binding specificity for il-4 and/or il-13.
This patent application is currently assigned to Domants Limited. Invention is credited to Steve Holmes, Kevin Moulder, Ian M. Tomlinson.
Application Number | 20070104710 11/338863 |
Document ID | / |
Family ID | 56290774 |
Filed Date | 2007-05-10 |
United States Patent
Application |
20070104710 |
Kind Code |
A1 |
Tomlinson; Ian M. ; et
al. |
May 10, 2007 |
Ligand that has binding specificity for IL-4 and/or IL-13
Abstract
The invention provides a dual-specific ligand comprising a first
immunoglobulin variable domain having a first binding specificity
and a complementary or non-complementary immunoglobulin variable
domain having a second binding specificity.
Inventors: |
Tomlinson; Ian M.;
(Cambridge, GB) ; Holmes; Steve; (Cambridge,
GB) ; Moulder; Kevin; (Cambridge, GB) |
Correspondence
Address: |
HAMILTON, BROOK, SMITH & REYNOLDS, P.C.
530 VIRGINIA ROAD
P.O. BOX 9133
CONCORD
MA
01742-9133
US
|
Assignee: |
Domants Limited
Cambridge
GB
|
Family ID: |
56290774 |
Appl. No.: |
11/338863 |
Filed: |
January 24, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11023959 |
Dec 28, 2004 |
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11338863 |
Jan 24, 2006 |
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PCT/GB03/02804 |
Jun 30, 2003 |
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11023959 |
Dec 28, 2004 |
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PCT/GB02/03014 |
Jun 28, 2002 |
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PCT/GB03/02804 |
Jun 30, 2003 |
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Current U.S.
Class: |
424/145.1 ;
530/388.23 |
Current CPC
Class: |
C07K 2317/92 20130101;
C07K 2317/622 20130101; C12N 2799/021 20130101; C07K 16/18
20130101; C07K 2317/31 20130101; C07K 16/40 20130101; C07K 2317/56
20130101; C07K 2317/21 20130101; C07K 16/241 20130101; A61K 47/60
20170801; C07K 2317/55 20130101; C07K 16/2878 20130101; C07K
2317/34 20130101; C07K 2317/569 20130101; A61K 2039/505 20130101;
C07K 16/2866 20130101 |
Class at
Publication: |
424/145.1 ;
530/388.23 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C07K 16/24 20060101 C07K016/24 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 28, 2003 |
GB |
GB 0327706.8 |
Dec 27, 2002 |
GB |
GB 0230202.4 |
Claims
1. A dual-specific ligand comprising a first immunoglobulin single
variable domain having binding specificity for IL-4 and a second
immunoglobulin single variable domain having binding specificity
for a second antigen or epitope, wherein said first single
immunoglobulin variable domain and said second immunoglobulin
single variable domain are mutually complementary and have
different binding specificities.
2. The dual-specific ligand of claim 1, wherein said first
immunoglobulin single variable domain is a V.sub.H domain and said
second immunoglobulin single variable domain is a V.sub.L domain,
or said first immunoglobulin single variable domain is a V.sub.L
domain and said second immunoglobulin single variable domain is a
V.sub.H domain.
3. An IgG comprising the dual-specific ligand of claim 1.
4. A method for treating allergic hypersensitivity in a mammal,
comprising administering a therapeutically effective dose of a
ligand of claim 1 to said mammal.
5. A dual-specific ligand comprising a first immunoglobulin single
variable domain having binding specificity for IL-13 and a second
immunoglobulin single variable domain having binding specificity
for a second antigen or epitope, wherein said first single
immunoglobulin variable domain and said second immunoglobulin
single variable domain are mutually complementary and have
different binding specificities.
6. The dual-specific ligand of claim 5, wherein said first
immunoglobulin single variable domain is a V.sub.H domain and said
second immunoglobulin single variable domain is a V.sub.L domain;
or said first immunoglobulin single variable domain is a V.sub.L
domain and said second immunoglobulin single variable domain is a
V.sub.H domain.
7. An IgG comprising the dual-specific ligand of claim 6.
8. A method for treating allergic hypersensitivity in a mammal,
comprising administering a therapeutically effective dose of a
ligand of claim 6 to said mammal.
9. A dual-specific ligand comprising a first immunoglobulin single
variable domain having binding specificity for IL-4 and a second
immunoglobulin single variable domain having binding specificity
for IL-13, wherein said first single immunoglobulin variable domain
and said second complementary binding immunoglobulin single
variable domain are mutually complementary.
10. The dual-specific ligand of claim 9, wherein said first
immunoglobulin single variable domain is a V.sub.H domain and said
second immunoglobulin single variable domain is a V.sub.L domain;
or said first immunoglobulin single variable domain is a V.sub.L
domain and said second immunoglobulin single variable domain is a
V.sub.H domain.
11. An IgG comprising the dual-specific ligand of claim 9.
12. A method for treating allergic hypersensitivity in a mammal,
comprising administering a therapeutically effective dose of a
ligand of claim 9 to said mammal.
13. A dual specific ligand that has binding specificity for
interleukin-4 (IL-4) and interleukin-13 (IL-13) comprising (1) a
protein moiety with binding specificity for IL-4, wherein the
protein moiety comprises a domain that has a binding site with
binding specificity for IL-4 and an immunoglobulin constant domain
selected from the group consisting of a heavy chain C.sub.H1 domain
and a C.sub.L domain; and (2) a protein moiety that has binding
specificity for IL-13, wherein the protein moiety comprises a
domain that has a binding site with binding specificity for IL-13
and an immunoglobulin constant domain selected from the group
consisting of a heavy chain C.sub.H1 domain and a C.sub.L
domain.
14. The dual specific ligand of claim 13 wherein said domain that
has a binding site with binding specificity for IL-4 is an
immunoglobulin variable domain.
15. The dual specific ligand of claim 13 wherein said domain that
has a binding site with binding specificity for IL-13 is an
immunoglobulin variable domain.
16. The dual specific ligand of claim 13 wherein said domain that
has a binding site with binding specificity for IL-4 is a V.sub.H
domain, and said domain that has a binding site with binding
specificity for IL-13 is a V.sub.H domain.
17. The dual specific ligand of claim 13 wherein said domain that
has a binding site with binding specificity for IL-4 is a V.sub.L
domain, and said domain that has a binding site with binding
specificity for IL-13 is a V.sub.L domain.
18. The dual specific ligand of claim 13 wherein said domain that
has a binding site with binding specificity for IL-4 is a V.sub.H
domain and said domain that has a binding site with binding
specificity for IL-13 is a V.sub.L domain, or said domain that has
a binding site with binding specificity for IL-4 is a V.sub.L
domain and said domain that has a binding site with binding
specificity for IL-13 is a V.sub.H domain
19. An IgG comprising the dual-specific ligand of claim 13.
20. A method for treating allergic hypersensitivity in a mammal,
comprising administering a therapeutically effective dose of a
ligand of claim 13 to said mammal.
21. A method for treating allergic asthma in a mammal, comprising
administering a therapeutically effective dose of a ligand of claim
13 to said mammal.
Description
RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S. patent
application Ser. No. 11/023,959, filed Dec. 28, 2004, which is a
continuation of International Application No. PCT/GB2003/002804,
filed on Jun. 30, 2003, published in English, which is a
continuation-in-part of International Application No.
PCT/GB02/03014, which designated the United States and was filed on
Jun. 28, 2002, published in English. This application claims
priority under 35 U.S.C. .sctn.119 or 365 to United Kingdom
Application No. GB 0327706.8, filed Nov. 28, 2003, and United
Kingdom Application No. GB 0230202.4, filed Dec. 27, 2002.
[0002] The entire teachings of the above applications are
incorporated herein by reference.
BACKGROUND
[0003] The present invention relates to dual specific ligands. In
particular, the invention provides a method for the preparation of
dual-specific ligands comprising a first immunoglobulin single
variable domain binding to a first antigen or epitope, and a second
immunoglobulin single variable domain binding to a second antigen
or epitope. More particularly, the invention relates to
dual-specific ligands wherein binding to at least one of the first
and second antigens or epitopes acts to increase the half-life of
the ligand in vivo. Open and closed conformation ligands comprising
more than one binding specificity are described.
[0004] The antigen binding domain of an antibody comprises two
separate regions: a heavy chain variable domain (V.sub.H) and a
light chain variable domain (V.sub.L: which can be either
V.sub..kappa. or V.sub..lamda.). The antigen binding site itself is
formed by six polypeptide loops: three from V.sub.H domain (H1, H2
and H3) and three from V.sub.L domain (L1, L2 and L3). A diverse
primary repertoire of V genes that encode the V.sub.H and V.sub.L
domains is produced by the combinatorial rearrangement of gene
segments. The V.sub.H gene is produced by the recombination of
three gene segments, V.sub.H, D and J.sub.H. In humans, there are
approximately 51 functional V.sub.H segments (Cook and Tomlinson
(1995) Immunol Today, 16: 237), 25 functional D segments (Corbett
et al. (1997) J. Mol. Biol., 268: 69) and 6 functional J.sub.H
segments (Ravetch et al. (1981) Cell, 27: 583), depending on the
haplotype. The V.sub.H segment encodes the region of the
polypeptide chain which forms the first and second antigen binding
loops of the V.sub.H domain (H1 and H2), whilst the V.sub.H, D and
J.sub.H segments combine to form the third antigen binding loop of
the V.sub.H domain (H3). The V.sub.L gene is produced by the
recombination of only two gene segments, V.sub.L and J.sub.L. In
humans, there are approximately 40 functional V.sub..kappa.
segments (Schable and Zachau (1993) Biol. Chem. Hoppe-Seyler, 374:
1001), 31 functional V.sub..lamda. segments (Williams et al. (1996)
J. Mol. Biol., 264: 220; Kawasaki et al. (1997) Genome Res., 7:
250), 5 functional J.sub..kappa. segments (Hieter et al. (1982) J.
Biol: Chem., 257: 1516) and 4 functional J.sub..lamda. segments
(Vasicek and Leder (1990) J. Exp. Med., 172: 609), depending on the
haplotype. The V.sub.L segment encodes the region of the
polypeptide chain which forms the first and second antigen binding
loops of the V.sub.L domain (L1 and L2), whilst the V.sub.L and
J.sub.L segments combine to form the third antigen binding loop of
the V.sub.L domain (L3). Antibodies selected from this primary
repertoire are believed to be sufficiently diverse to bind almost
all antigens with at least moderate affinity. High affinity
antibodies are produced by "affinity maturation" of the rearranged
genes, in which point mutations are generated and selected by the
immune system on the basis of improved binding.
[0005] Analysis of the structures and sequences of antibodies has
shown that five of the six antigen binding loops (H1, H2, L1, L2,
L3) possess a limited number of main-chain conformations or
canonical structures (Chothia and Lesk (1987) J. Mol. Biol., 196:
901; Chothia et al. (1989) Nature, 342: 877). The main-chain
conformations are determined by (i) the length of the antigen
binding loop, and (ii) particular residues, or types of residue, at
certain key position in the antigen binding loop and the antibody
framework. Analysis of the loop lengths and key residues has
enabled us to the predict the main-chain conformations of H1, H2,
L1, L2 and L3 encoded by the majority of human antibody sequences
(Chothia et al. (1992) J. Mol. Biol., 227: 799; Tomlinson et al.
(1995) EMBO J., 14: 4628; Williams et al. (1996) J. Mol. Biol.,
264: 220). Although the H3 region is much more diverse in terms of
sequence, length and structure (due to the use of D segments), it
also forms a limited number of main-chain conformations for short
loop lengths which depend on the length and the presence of
particular residues, or types of residue, at key positions in the
loop and the antibody framework (Martin et al. (1996) J. Mol.
Biol., 263: 800; Shirai et al. (1996) FEBS Letters, 399: 1.
[0006] Bispecific antibodies comprising complementary pairs of
V.sub.H and V.sub.L regions are known in the art. These bispecific
antibodies must comprise two pairs of V.sub.H and V.sub.Ls, each
V.sub.H/V.sub.L pair binding to a single antigen or epitope.
Methods described involve hybrid hybridomas (Milstein & Cuello
A C, Nature 305:537-40), minibodies (Hu et al., (1996) Cancer Res
56:3055-3061), diabodies (Holliger et al., (1993) Proc. Natl. Acad.
Sci. USA 90, 6444-6448; WO 94/13804), chelating recombinant
antibodies (CRAbs; (Neri et al., (1995) J. Mol. Biol. 246,
367-373), biscFv (e.g. Atwell et al., (1996) Mol. Immunol. 33,
1301-1312), "knobs in holes" stabilised antibodies (Carter et al.,
(1997) Protein Sci. 6, 781-788). In each case each antibody species
comprises two antigen-binding sites, each fashioned by a
complementary pair of V.sub.H and V.sub.L domains. Each antibody is
thereby able to bind to two different antigens or epitopes at the
same time, with the binding to EACH antigen or epitope mediated by
a V.sub.H and its complementary V.sub.L domain. Each of these
techniques presents its particular disadvantages; for instance in
the case of hybrid hybridomas, inactive V.sub.H/V.sub.L pairs can
greatly reduce the fraction of bispecific IgG. Furthermore, most
bispecific approaches rely on the association of the different
V.sub.H/V.sub.L pairs or the association of V.sub.H and V.sub.L
chains to recreate the two different V.sub.H/V.sub.L binding sites.
It is therefore impossible to control the ratio of binding sites to
each antigen or epitope in the assembled molecule and thus many of
the assembled molecules will bind to one antigen or epitope but not
the other. In some cases it has been possible to engineer the heavy
or light chains at the sub-unit interfaces (Carter et al., 1997) in
order to improve the number of molecules which have binding sites
to both antigens or epitopes but this never results in all
molecules having binding to both antigens or epitopes.
[0007] There is some evidence that two different antibody binding
specificities might be incorporated into the same binding site, but
these generally represent two or more specificities that correspond
to structurally related antigens or epitopes or to antibodies that
are broadly cross-reactive. For example, cross-reactive antibodies
have been described, usually where the two antigens are related in
sequence and structure, such as hen egg white lysozyme and turkey
lysozyme (McCafferty et al., WO 92/01047) or to free hapten and to
hapten conjugated to carrier (Griffiths A D et al. EMBO J 1994
13:14 3245-60). In a further example, WO 02/02773 (Abbott
Laboratories) describes antibody molecules with "dual specificity".
The antibody molecules referred to are antibodies raised or
selected against multiple antigens, such that their specificity
spans more than a single antigen. Each complementary
V.sub.H/V.sub.L pair in the antibodies of WO 02/02773 specifies a
single binding specificity for two or more structurally related
antigens; the V.sub.H and V.sub.L domains in such complementary
pairs do not each possess a separate specificity. The antibodies
thus have a broad single specificity which encompasses two
antigens, which are structurally related. Furthermore natural
autoantibodies have been described that are polyreactive (Casali
& Notkins, Ann. Rev. Immunol. 7, 515-531), reacting with at
least two (usually more) different antigens or epitopes that are
not structurally related. It has also been shown that selections of
random peptide repertoires using phage display technology on a
monoclonal antibody will identify a range of peptide sequences that
fit the antigen binding site. Some of the sequences are highly
related, fitting a consensus sequence, whereas others are very
different and have been termed mimotopes (Lane & Stephen,
Current Opinion in Immunology, 1993, 5, 268-271). It is therefore
clear that a natural four-chain antibody, comprising associated and
complementary V.sub.H and V.sub.L domains, has the potential to
bind to many different antigens from a large universe of known
antigens. It is less clear how to create a binding site to two
given antigens in the same antibody, particularly those which are
not necessarily structurally related.
[0008] Protein engineering methods have been suggested that may
have a bearing on this. For example it has also been proposed that
a catalytic antibody could be created with a binding activity to a
metal ion through one variable domain, and to a hapten (substrate)
through contacts with the metal ion and a complementary variable
domain (Barbas et al., 1993 Proc. Natl. Acad. Sci USA 90,
6385-6389). However in this case, the binding and catalysis of the
substrate (first antigen) is proposed to require the binding of the
metal ion (second antigen). Thus the binding to the V.sub.H/V.sub.L
pairing relates to a single but multi-component antigen.
[0009] Methods have been described for the creation of bispecific
antibodies from camel antibody heavy chain single domains in which
binding contacts for one antigen are created in one variable
domain, and for a second antigen in a second variable domain.
However the variable domains were not complementary. Thus a first
heavy chain variable domain is selected against a first antigen,
and a second heavy chain variable domain against a second antigen,
and then both domains are linked together on the same chain to give
a bispecific antibody fragment (Conrath et al., J. Biol. Chem. 270,
27589-27594). However the camel heavy chain single domains are
unusual in that they are derived from natural camel antibodies
which have no light chains, and indeed the heavy chain single
domains are unable to associate with camel light chains to form
complementary V.sub.H and V.sub.L pairs.
[0010] Single heavy chain variable domains have also been
described, derived from natural antibodies which are normally
associated with light chains (from monoclonal antibodies or from
repertoires of domains; see EP-A-0368684). These heavy chain
variable domains have been shown to interact specifically with one
or more related antigens but have not been combined with other
heavy or light chain variable domains to create a ligand with a
specificity for two or more different antigens. Furthermore, these
single domains have been shown to have a very short in vivo
half-life. Therefore such domains are of limited therapeutic
value.
[0011] It has been suggested to make bispecific antibody fragments
by linking heavy chain variable domains of different specificity
together (as described above). The disadvantage with this approach
is that isolated antibody variable domains may have a hydrophobic
interface that normally makes interactions with the light chain and
is exposed to solvent and may be "sticky" allowing the single
domain to bind to hydrophobic surfaces. Furthermore, in the absence
of a partner light chain the combination of two or more different
heavy chain variable domains and their association, possibly via
their hydrophobic interfaces, may prevent them from binding to one
in not both of the ligands they are able to bind in isolation.
Moreover, in this case the heavy chain variable domains would not
be associated with complementary light chain variable domains and
thus may be less stable and readily unfold (Worn & Pluckthun,
1998 Biochemistry 37, 13120-7).
SUMMARY OF THE INVENTION
[0012] The inventors have described, in their copending
international patent application WO 03/002609 as well as copending
unpublished UK patent application 0230203.2, dual specific
immunoglobulin ligands which comprise immunoglobulin single
variable domains which each have different specificities. The
domains may act in competition with each other or independently to
bind antigens or epitopes on target molecules.
[0013] The invention relates to a dual-specific ligand comprising a
first immunoglobulin single variable domain having binding
specificity for IL-4 and a second immunoglobulin single variable
domain having binding specificity for a second antigen or epitope,
wherein said first single immunoglobulin variable domain and said
second immunoglobulin single variable domain are mutually
complementary and have different binding specificities. The first
immunoglobulin single variable domain can be a V.sub.H domain and
said second immunoglobulin single variable domain can be a V.sub.L
domain, or said first immunoglobulin single variable domain can be
a V.sub.L domain and said second immunoglobulin single variable
domain can be a V.sub.H domain.
[0014] The invention relates to a dual-specific ligand comprising a
first immunoglobulin single variable domain having binding
specificity for IL-13 and a second immunoglobulin single variable
domain having binding specificity for a second antigen or epitope,
wherein said first single immunoglobulin variable domain and said
second immunoglobulin single variable domain are mutually
complementary and have different binding specificities. The first
immunoglobulin single variable domain can be a V.sub.H domain and
said second immunoglobulin single variable domain can be a V.sub.L
domain, or said first immunoglobulin single variable domain can be
a V.sub.L domain and said second immunoglobulin single variable
domain can be a V.sub.H domain.
[0015] In some embodiments, the dual-specific ligand comprises a
first immunoglobulin single variable domain having binding
specificity for IL-4 and a second immunoglobulin single variable
domain having binding specificity for IL-13, wherein said first
single immunoglobulin variable domain and said second complementary
binding immunoglobulin single variable domain are mutually
complementary. The first immunoglobulin single variable domain can
be a V.sub.H domain and said second immunoglobulin single variable
domain can be a V.sub.L domain, or said first immunoglobulin single
variable domain can be a V.sub.L domain and said second
immunoglobulin single variable domain can be a V.sub.H domain.
[0016] The invention relates to an IgG comprising a dual-specific
ligand that binds IL-4, IL-13 or IL-4 and IL-13 of the
invention.
[0017] The invention relates to a method for treating allergic
hypersensitivity in a mammal, comprising administering a
therapeutically effective dose of a dual specific ligand that binds
IL-4, IL-13 or IL-4 and IL-13 to said mammal.
[0018] The invention relates to a dual specific ligand that has
binding specificity for interleukin-4 (IL-4) and interleukin-13
(IL-13) comprising (1) a protein moiety with binding specificity
for IL-4, wherein the protein moiety comprises a domain that has a
binding site with binding specificity for IL-4 and an
immunoglobulin constant domain selected from the group consisting
of a heavy chain C.sub.H1 domain and a C.sub.L domain; and (2) a
protein moiety that has binding specificity for IL-13, wherein the
protein moiety comprises a domain that has a binding site with
binding specificity for IL-13 and an immunoglobulin constant domain
selected from the group consisting of a heavy chain C.sub.H1 domain
and a C.sub.L domain. The domain that has a binding site with
binding specificity for IL-4 can be an immunoglobulin variable
domain. The domain that has a binding site with binding specificity
for IL-13 can be an immunoglobulin variable domain. In some
embodiments, the domain that has a binding site with binding
specificity for IL-4 is a V.sub.H domain, and the domain that has a
binding site with binding specificity for IL-13 is a V.sub.H
domain. In other embodiments, the domain that has a binding site
with binding specificity for IL-4 is a V.sub.L domain, and said
domain that has a binding site with binding specificity for IL-13
is a V.sub.L domain. In particular embodiments, the domain that has
a binding site with binding specificity for IL-4 is a V.sub.H
domain and said domain that has a binding site with binding
specificity for IL-13 is a V.sub.L domain, or said domain that has
a binding site with binding specificity for IL-4 is a V.sub.L
domain and said domain that has a binding site with binding
specificity for IL-13 is a V.sub.H domain
[0019] The invention relates to an IgG comprising a dual-specific
ligand that binds IL-4 and IL-13 the invention.
[0020] The invention relates to a method for treating allergic
hypersensitivity in a mammal, comprising administering a
therapeutically effective dose of a dual specific ligand that binds
IL-4 and IL-13 to said mammal.
[0021] The invention relates to a method for treating allergic
asthma in a mammal, comprising administering a therapeutically
effective dose of a dual specific ligand that binds IL-4 and IL-13
to said mammal.
[0022] In a first configuration, the present invention provides a
further improvement in dual specific ligands as developed by the
present inventors, in which one specificity of the ligand is
directed towards a protein or polypeptide present in vivo in an
organism which can act to increase the half-life of the ligand by
binding to it.
[0023] Accordingly, in a first aspect, there is provided a
dual-specific ligand comprising a first immunoglobulin single
variable domain having a binding specificity to a first antigen or
epitope and a second complementary immunoglobulin single variable
domain having a binding activity to a second antigen or epitope,
wherein one or both of said antigens or epitopes acts to increase
the half-life of the ligand in vivo and wherein said first and
second domains lack mutually complementary domains which share the
same specificity, provided that said dual specific ligand does not
consist of an anti-HSA V.sub.H domain and an anti-.beta.
galactosidase V.sub..kappa. domain. Preferably, that neither of the
first or second variable domains binds to human serum albumin
(HSA).
[0024] Antigens or epitopes which increase the half-life of a
ligand as described herein are advantageously present on proteins
or polypeptides found in an organism in vivo. Examples include
extracellular matrix proteins, blood proteins, and proteins present
in various tissues in the organism. The proteins act to reduce the
rate of ligand clearance from the blood, for example by acting as
bulking agents, or by anchoring the ligand to a desired site of
action. Examples of antigens/epitopes which increase half-life in
vivo are given in Annex 1 below.
[0025] Increased half-life is useful in in vivo applications of
immunoglobulins, especially antibodies and most especially antibody
fragments of small size. Such fragments (Fvs, disulphide bonded
Fvs, Fabs, scFvs, dAbs) suffer from rapid clearance from the body;
thus, whilst they are able to reach most parts of the body rapidly,
and are quick to produce and easier to handle, their in vivo
applications have been limited by their only brief persistence in
vivo. The invention solves this problem by providing increased
half-life of the ligands in vivo and consequently longer
persistence times in the body of the functional activity of the
ligand.
[0026] Methods for pharmacokinetic analysis and determination of
ligand half-life will be familiar to those skilled in the art.
Details may be found in Kenneth, A et al: Chemical Stability of
Pharmaceuticals: A Handbook for Pharmacists and in Peters et al,
Pharmacokinetc analysis: A Practical Approach (1996). Reference is
also made to "Pharmacokinetics", M Gibaldi & D Perron,
published by Marcel Dekker, 2.sup.nd Rev. ex edition (1982), which
describes pharmacokinetic parameters such as t alpha and t beta
half lives and area under the curve (AUC).
[0027] Half lives (t1/2 alpha and t1/2 beta) and AUC can be
determined from a curve of serum concentration of ligand against
time. The WinNonlin analysis package (available from Pharsight
Corp., Mountain View, Calif. 94040, USA) can be used, for example,
to model the curve. In a first phase (the alpha phase) the ligand
is undergoing mainly distribution in the patient, with some
elimination. A second phase (beta phase) is the terminal phase when
the ligand has been distributed and the serum concentration is
decreasing as the ligand is cleared from the patient. The t alpha
half life is the half life of the first phase and the t beta half
life is the half life of the second phase. Thus, advantageously,
the present invention provides a ligand or a composition comprising
a ligand according to the invention having a t.alpha. half-life in
the range of 15 minutes or more. In one embodiment, the lower end
of the range is 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4
hours, 5 hours, 6 hours, 7 hours, 10 hours, 11 hours or 12 hours.
In addition, or alternatively, a ligand or composition according to
the invention will have a t.alpha. half life in the range of up to
and including 12 hours. In one embodiment, the upper end of the
range is 11, 10, 9, 8, 7, 6 or 5 hours. An example of a suitable
range is 1 to 6 hours, 2 to 5 hours or 3 to 4 hours.
[0028] Advantageously, the present invention provides a ligand or a
composition comprising a ligand according to the invention having a
t.beta. half-life in the range of 2.5 hours or more. In one
embodiment, the lower end of the range is 3 hours, 4 hours, 5
hours, 6 hours, 7 hours, 10 hours, 11 hours, or 12 hours. In
addition, or alternatively, a ligand or composition according to
the invention has a t.beta. half-life in the range of up to and
including 21 days. In one embodiment, the upper end of the range is
12 hours, 24 hours, 2 days, 3 days, 5 days, 10 days, 15 days or 20
days. Advantageously a ligand or composition according to the
invention will have a t.beta. half life in the range 12 to 60
hours. In a further embodiment, it will be in the range 12 to 48
hours. In a further embodiment still, it will be in the range 12 to
26 hours.
[0029] In addition, or alternatively to the above criteria, the
present invention provides a ligand or a composition comprising a
ligand according to the invention having an AUC value (area under
the curve) in the range of 1 mg.min/ml or more. In one embodiment,
the lower end of the range is 5, 10, 15, 20, 30, 100, 200 or 300
mg.min/ml. In addition, or alternatively, a ligand or composition
according to the invention has an AUC in the range of up to 600
mg.min/ml. In one embodiment, the upper end of the range is 500,
400, 300, 200, 150, 100, 75 or 50 mg.min/ml. Advantageously a
ligand according to the invention will have a AUC in the range
selected from the group consisting of the following: 15 to 150
mg.min/ml, 15 to 100 mg.min/ml, 15 to 75 mg.min/ml, and 15 to 50
mg.min/ml.
[0030] In a first embodiment, the dual specific ligand comprises
two complementary variable domains, i.e. two variable domains that,
in their natural environment, are capable of operating together as
a cognate pair or group even if in the context of the present
invention they bind separately to their cognate epitopes. For
example, the complementary variable domains may be immunoglobulin
heavy chain and light chain variable domains (V.sub.H and V.sub.L).
V.sub.H and V.sub.L domains are advantageously provided by scFv or
Fab antibody fragments. Variable domains may be linked together to
form multivalent ligands by, for example: provision of a hinge
region at the C-terminus of each V domain and disulphide bonding
between cysteines in the hinge regions; or provision of dAbs each
with a cysteine at the C-terminus of the domain, the cysteines
being disulphide bonded together; or production of V-CH & V-CL
to produce a Fab format; or use of peptide linkers (for example
Gly.sub.4Ser linkers discussed hereinbelow) to produce dimers,
trimers and further multimers.
[0031] The inventors have found that the use of complementary
variable domains allows the two domain surfaces to pack together
and be sequestered from the solvent. Furthermore the complementary
domains are able to stabilise each other. In addition, it allows
the creation of dual-specific IgG antibodies without the
disadvantages of hybrid hybridomas as used in the prior art, or the
need to engineer heavy or light chains at the sub-unit interfaces.
The dual-specific ligands of the first aspect of the present
invention have at least one V.sub.H/V.sub.L pair. A bispecific IgG
according to this invention will therefore comprise two such pairs,
one pair on each arm of the Y-shaped molecule. Unlike conventional
bispecific antibodies or diabodies, therefore, where the ratio of
chains used is determinative in the success of the preparation
thereof and leads to practical difficulties, the dual specific
ligands of the invention are free from issues of chain balance.
Chain imbalance in conventional bi-specific antibodies results from
the association of two different V.sub.L chains with two different
V.sub.H chains, where V.sub.L chain 1 together with V.sub.H chain 1
is able to bind to antigen or epitope 1 and V.sub.L chain 2
together with V.sub.H chain 2 is able to bind to antigen or epitope
2 and the two correct pairings are in some way linked to one
another. Thus, only when V.sub.L chain 1 is paired with V.sub.H
chain 1 and V.sub.L chain 2 is paired with V.sub.H chain 2 in a
single molecule is bi-specificity created. Such bi-specific
molecules can be created in two different ways. Firstly, they can
be created by association of two existing V.sub.H/V.sub.L pairings
that each bind to a different antigen or epitope (for example, in a
bi-specific IgG). In this case the V.sub.H/V.sub.L pairings must
come all together in a 1:1 ratio in order to create a population of
molecules all of which are bi-specific. This never occurs (even
when complementary CH domain is enhanced by "knobs into holes"
engineering) leading to a mixture of bi-specific molecules and
molecules that are only able to bind to one antigen or epitope but
not the other. The second way of creating a bi-specific antibody is
by the simultaneous association of two different V.sub.H chain with
two different V.sub.L chains (for example in a bi-specific
diabody). In this case, although there tends to be a preference for
V.sub.L chain 1 to pair with V.sub.H chain 1 and V.sub.L chain 2 to
pair with V.sub.H chain 2 (which can be enhanced by "knobs into
holes" engineering of the V.sub.L and V.sub.H domains), this paring
is never achieved in all molecules, leading to a mixed formulation
whereby incorrect pairings occur that are unable to bind to either
antigen or epitope.
[0032] Bi-specific antibodies constructed according to the
dual-specific ligand approach according to the first aspect of the
present invention overcome all of these problems because the
binding to antigen or epitope 1 resides within the V.sub.H or
V.sub.L domain and the binding to antigen or epitope 2 resides with
the complementary V.sub.L or V.sub.H domain, respectively. Since
V.sub.H and V.sub.L domains pair on a 1:1 basis all V.sub.H/V.sub.L
pairings will be bi-specific and thus all formats constructed using
these V.sub.H/V.sub.L pairings (Fv, scFvs, Fabs, minibodies, IgGs
etc) will have 100% bi-specific activity.
[0033] In the context of the present invention, first and second
"epitopes" are understood to be epitopes which are not the same and
are not bound by a single monospecific ligand. In the first
configuration of the invention, they are advantageously on
different antigens, one of which acts to increase the half-life of
the ligand in vivo. Likewise, the first and second antigens are
advantageously not the same.
[0034] The dual specific ligands of the invention do not include
ligands as described in WO 02/02773. Thus, the ligands of the
present invention do not comprise complementary V.sub.H/V.sub.L
pairs which bind any one or more antigens or epitopes
co-operatively. Instead, the ligands according to the first aspect
of the invention comprise a V.sub.H/V.sub.L complementary pair,
wherein the V domains have different specificities.
[0035] Moreover, the ligands according to the first aspect of the
invention comprise V.sub.H/V.sub.L complementary pairs having
different specificities for non-structurally related epitopes or
antigens. Structurally related epitopes or antigens are epitopes or
antigens which possess sufficient structural similarity to be bound
by a conventional V.sub.H/V.sub.L complementary pair which acts in
a co-operative manner to bind an antigen or epitope; in the case of
structurally related epitopes, the epitopes are sufficiently
similar in structure that they "fit" into the same binding pocket
formed at the antigen binding site of the V.sub.H/V.sub.L
dimer.
[0036] In a second aspect, the present invention provides a ligand
comprising a first immunoglobulin variable domain having a first
antigen or epitope binding specificity and a second immunoglobulin
variable domain having a second antigen or epitope binding
specificity wherein one or both of said first and second variable
domains bind to an antigen which increases the half-life of the
ligand in vivo, and the variable domains are not complementary to
one another.
[0037] In one embodiment, binding to one variable domain modulates
the binding of the ligand to the second variable domain.
[0038] In this embodiment, the variable domains may be, for
example, pairs of V.sub.H domains or pairs of V.sub.L domains.
Binding of antigen at the first site may modulate, such as enhance
or inhibit, binding of an antigen at the second site. For example,
binding at the first site at least partially inhibits binding of an
antigen at a second site. In such an embodiment, the ligand may for
example be maintained in the body of a subject organism in vivo
through binding to a protein which increases the half-life of the
ligand until such a time as it becomes bound to the second target
antigen and dissociates from the half-life increasing protein.
[0039] Modulation of binding in the above context is achieved as a
consequence of the structural proximity of the antigen binding
sites relative to one another. Such structural proximity can be
achieved by the nature of the structural components linking the two
or more antigen binding sites, eg by the provision of a ligand with
a relatively rigid structure that holds the antigen binding sites
in close proximity. Advantageously, the two or more antigen binding
sites are in physically close proximity to one another such that
one site modulates the binding of antigen at another site by a
process which involves steric hindrance and/or conformational
changes within the immunoglobulin molecule.
[0040] The first and the second antigen binding domains may be
associated either covalently or non-covalently. In the case that
the domains are covalently associated, then the association may be
mediated for example by disulphide bonds or by a polypeptide linker
such as (Gly.sub.4Ser).sub.n, where n=from 1 to 8, eg, 2, 3, 4, 5
or 7.
[0041] Ligands according to the invention may be combined into
non-immunoglobulin multi-ligand structures to form multivalent
complexes, which bind target molecules with the same antigen,
thereby providing superior avidity, while at least one variable
domain binds an antigen to increase the half life of the multimer.
For example natural bacterial receptors such as SpA have been used
as scaffolds for the grafting of CDRs to generate ligands which
bind specifically to one or more epitopes. Details of this
procedure are described in U.S. Pat. No. 5,831,012. Other suitable
scaffolds include those based on fibronectin and affibodies.
Details of suitable procedures are described in WO 98/58965. Other
suitable scaffolds include lipocallin and CTLA4, as described in
van den Beuken et al., J. Mol. Biol. (2001) 310, 591-601, and
scaffolds such as those described in WO0069907 (Medical Research
Council), which are based for example on the ring structure of
bacterial GroEL or other chaperone polypeptides.
[0042] Protein scaffolds may be combined; for example, CDRs may be
grafted on to a CTLA4 scaffold and used together with
immunoglobulin V.sub.H or V.sub.L domains to form a ligand.
Likewise, fibronectin, lipocallin and other scaffolds may be
combined.
[0043] In the case that the variable domains are selected from
V-gene repertoires selected for instance using phage display
technology as herein described, then these variable domains can
comprise a universal framework region, such that is they may be
recognised by a specific generic ligand as herein defined. The use
of universal frameworks, generic ligands and the like is described
in WO99/20749. In the present invention, reference to phage display
includes the use of both phage and/or phagemids.
[0044] Where V-gene repertoires are used variation in polypeptide
sequence is preferably located within the structural loops of the
variable domains. The polypeptide sequences of either variable
domain may be altered by DNA shuffling or by mutation in order to
enhance the interaction of each variable domain with its
complementary pair.
[0045] In a preferred embodiment of the invention the
`dual-specific ligand` is a single chain Fv fragment. In an
alternative embodiment of the invention, the `dual-specific ligand`
consists of a Fab region of an antibody. The term "Fab region"
includes a Fab-like region where two VH or two VL domains are
used.
[0046] The variable regions may be derived from antibodies directed
against target antigens or epitopes. Alternatively they may be
derived from a repertoire of single antibody domains such as those
expressed on the surface of filamentous bacteriophage. Selection
may be performed as described below.
[0047] In a third aspect, the invention provides a method for
producing a ligand comprising a first immunoglobulin single
variable domain having a first binding specificity and a second
single immunoglobulin single variable domain having a second
(different) binding specificity, one or both of the binding
specificities being specific for an antigen which increases the
half-life of the ligand in vivo, the method comprising the steps
of:
[0048] (a) selecting a first variable domain by its ability to bind
to a first epitope,
[0049] (b) selecting a second variable region by its ability to
bind to a second epitope,
[0050] (c) combining the variable domains; and
[0051] (d) selecting the ligand by its ability to bind to said
first epitope and to said second epitope.
[0052] The ligand can bind to the first and second epitopes either
simultaneously or, where there is competition between the binding
domains for epitope binding, the binding of one domain may preclude
the binding of another domain to its cognate epitope. In one
embodiment, therefore, step (d) above requires simultaneous binding
to both first and second (and possibly further) epitopes; in
another embodiment, the binding to the first and second epitoes is
not simultaneous.
[0053] The epitopes are preferably on separate antigens.
[0054] Ligands advantageously comprise V.sub.H/V.sub.L
combinations, or V.sub.H/V.sub.H or V.sub.L/V.sub.L combinations of
immunoglobulin variable domains, as described above. The ligands
may moreover comprise camelid V.sub.HH domains, provided that the
V.sub.HH domain which is specific for an antigen which increases
the half-life of the ligand in vivo does not bind Hen egg white
lysozyme (HEL), porcine pancreatic alpha-amylase or NmC-A; hcg,
BSA-linked RR6 azo dye or S. mutans HG982 cells, as described in
Conrath et al., (2001) JBC 276:7346-7350 and WO99/23221, neither of
which describe the use of a specificity for an antigen which
increases half-life to increase the half life of the ligand in
vivo.
[0055] In one embodiment, said first variable domain is selected
for binding to said first epitope in absence of a complementary
variable domain. In a further embodiment, said first variable
domain is selected for binding to said first epitope/antigen in the
presence of a third variable domain in which said third variable
domain is different from said second variable domain and is
complementary to the first domain. Similarly, the second domain may
be selected in the absence or presence of a complementary variable
domain.
[0056] The antigens or epitopes targeted by the ligands of the
invention, in addition to the half-life enhancing protein, may be
any antigen or epitope but advantageously is an antigen or epitope
that is targeted with therapeutic benefit. The invention provides
ligands, including open conformation, closed conformation and
isolated dAb monomer ligands, specific for any such target,
particularly those targets further identified herein. Such targets
may be, or be part of, polypeptides, proteins or nucleic acids,
which may be naturally occurring or synthetic. In this respect, the
ligand of the invention may bind the epiotpe or antigen and act as
an antagonist or agonist (eg, EPO receptor agonist). One skilled in
the art will appreciate that the choice is large and varied. They
may be for instance human or animal proteins, cytokines, cytokine
receptors, enzymes co-factors for enzymes or DNA binding proteins.
Suitable cytokines and growth factors include but are not limited
to: ApoE, Apo-SAA, BDNF, Cardiotrophin-1, EGF, EGF receptor,
ENA-78, Eotaxin, Eotaxin-2, Exodus-2, EpoR, FGF-acidic, FGF-basic,
fibroblast growth factor-10, FLT3 ligand, Fractalkine (CX3C), GDNF,
G-CSF, GM-CSF, GF-.beta.1, insulin, IFN-.gamma., IGF-I, IGF-II,
IL-1.alpha., IL-1.beta., IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8
(72 a.a.), IL-8 (77 a.a.), IL-9, IL-10, IL-11, IL-12, IL-13, IL-15,
IL-16, IL-17, IL-18 (IGIF), Inhibin .alpha., Inhibin .beta., IP-10,
keratinocyte growth factor-2 (KGF-2), KGF, Leptin, LIF,
Lymphotactin, Mullerian inhibitory substance, monocyte colony
inhibitory factor, monocyte attractant protein, M-CSF, MDC (67
a.a.), MDC (69 a.a.), MCP-1 (MCAF), MCP-2, MCP-3, MCP-4, MDC (67
a.a.), MDC (69 a.a.), MIG, MIP-1.alpha., MIP-1.beta., MIP-3.alpha.,
MIP-3.beta., MIP-4, myeloid progenitor inhibitor factor-1 (MPIF-1),
NAP-2, Neurturin, Nerve growth factor, .beta.-NGF, NT-3, NT-4,
Oncostatin M, PDGF-AA, PDGF-AB, PDGF-BB, PF-4, RANTES, SDF1.alpha.,
SDF1.beta., SCF, SCGF, stem cell factor (SCF), TARC, TGF-.alpha.,
TGF-.beta., TGF-.beta.2, TGF-.beta.3, tumour necrosis factor (TNF),
TNF-.alpha., TNF-.beta., TNF receptor I, TNF receptor II, TNIL-1,
TPO, VEGF, VEGF receptor 1, VEGF receptor 2, VEGF receptor 3,
GCP-2, GRO/MGSA, GRO-.beta., GRO-.gamma., HCC1, 1-309, HER 1, HER
2, HER 3 and HER 4. Cytokine receptors include receptors for the
foregoing cytokines. It will be appreciated that this list is by no
means exhaustive.
[0057] In one embodiment of the invention, the variable domains are
derived from a respective antibody directed against the antigen or
epitope. In a preferred embodiment the variable domains are derived
from a repertoire of single variable antibody domains.
[0058] In a further aspect, the present invention provides one or
more nucleic acid molecules encoding at least a dual-specific
ligand as herein defined. The dual specific ligand may be encoded
on a single nucleic acid molecule; alternatively, each domain may
be encoded by a separate nucleic acid molecule. Where the ligand is
encoded by a single nucleic acid molecule, the domains may be
expressed as a fusion polypeptide, in the manner of a scFv
molecule, or may be separately expressed and subsequently linked
together, for example using chemical linking agents. Ligands
expressed from separate nucleic acids will be linked together by
appropriate means.
[0059] The nucleic acid may further encode a signal sequence for
export of the polypeptides from a host cell upon expression and may
be fused with a surface component of a filamentous bacteriophage
particle (or other component of a selection display system) upon
expression.
[0060] In a further aspect the present invention provides a vector
comprising nucleic acid encoding a dual specific ligand according
to the present invention.
[0061] In a yet further aspect, the present invention provides a
host cell transfected with a vector encoding a dual specific ligand
according to the present invention.
[0062] Expression from such a vector may be configured to produce,
for example on the surface of a bacteriophage particle, variable
domains for selection. This allows selection of displayed variable
regions and thus selection of `dual-specific ligands` using the
method of the present invention.
[0063] The present invention further provides a kit comprising at
least a dual-specific ligand according to the present
invention.
[0064] Dual-Specific ligands according to the present invention
preferably comprise combinations of heavy and light chain domains.
For example, the dual specific ligand may comprise a V.sub.H domain
and a V.sub.L domain, which may be linked together in the form of
an scFv. In addition, the ligands may comprise one or more C.sub.H
or C.sub.L domains. For example, the ligands may comprise a
C.sub.H1 domain, C.sub.H2 or C.sub.H3 domain, and/or a C.sub.L
domain, C.mu.1, C.mu.2, C.mu.3 or C.mu.4 domains, or any
combination thereof. A hinge region domain may also be included.
Such combinations of domains may, for example, mimic natural
antibodies, such as IgG or IgM, or fragments thereof, such as Fv,
scFv, Fab or F(ab').sub.2 molecules. Other structures, such as a
single arm of an IgG molecule comprising V.sub.H, V.sub.L, C.sub.H1
and C.sub.L domains, are envisaged.
[0065] The dual specific ligand can comprise a heavy chain constant
region of an immunoglobulin (e.g., IgG (e.g., IgG1, IgG2, IgG3,
IgG4) IgM, IgA, IgD or IgE) or portoion thereof (e.g., Fc portion)
and/or a light chain constant region (e.g., C.sub..lamda.,
C.sub..kappa.). For example, the ligand can comprise CH1 of IgG1
(e.g., human IgG1), CH1 and CH2 of IgG1 (e.g., human IgG1), CH1,
CH2 and CH3 of IgG1 (e.g., human IgG1), CH2 and CH3 of IgG1 (e.g.,
human IgG1), or CH1 and CH3 of IgG1 (e.g., human IgG1). A hinge
region domain may also be included. Such combinations of domains
may, for example, mimic natural antibodies, such as IgG or IgM, or
fragments thereof, such as Fv, scFv, Fab or F(ab').sub.2 molecules.
In addition, a particular constant region or Fc portion (e.g.,
constant region or Fc portion of an IgG, such as IgG1 (e.g., CH1,
CH2 and CH3; CH2 and CH3)), variant or portion thereof can be
selected in order to tailor effector function. For example, if
complement activation and/or antibody dependent cellular
cytotoxicity (ADCC) function is desired, the ligand can be an
IgG1-like format. If desired, the IgG-like format can comprise a
mutated constant region (variant IgG heavy chain constant region)
to minimize binding to Fc receptors and/or ability to fix
complement (see e.g. Winter et al, GB 2,209,757 B; Morrison et al.,
WO 89/07142; Morgan et al., WO 94/29351, Dec. 22, 1994).
[0066] In a preferred embodiment of the invention, the variable
regions are selected from single domain V gene repertoires.
Generally the repertoire of single antibody domains is displayed on
the surface of filamentous bacteriophage. In a preferred embodiment
each single antibody domain is selected by binding of a phage
repertoire to antigen.
[0067] In a preferred embodiment of the invention each single
variable domain may be selected for binding to its target antigen
or epitope in the absence of a complementary variable region. In an
alternative embodiment, the single variable domains may be selected
for binding to its target antigen or epitope in the presence of a
complementary variable region. Thus the first single variable
domain may be selected in the presence of a third complementary
variable domain, and the second variable domain may be selected in
the presence of a fourth complementary variable domain. The
complementary third or fourth variable domain may be the natural
cognate variable domain having the same specificity as the single
domain being tested, or a non-cognate complementary domain--such as
a "dummy" variable domain.
[0068] Preferably, the dual specific ligand of the invention
comprises only two variable domains although several such ligands
may be incorporated together into the same protein, for example two
such ligands can be incorporated into an IgG or a multimeric
immunoglobulin, such as IgM. Alternatively, in another embodiment a
plurality of dual specific ligands are combined to form a multimer.
For example, two different dual specific ligands are combined to
create a tetra-specific molecule.
[0069] It will be appreciated by one skilled in the art that the
light and heavy variable regions of a dual-specific ligand produced
according to the method of the present invention may be on the same
polypeptide chain, or alternatively, on different polypeptide
chains. In the case that the variable regions are on different
polypeptide chains, then they may be linked via a linker, generally
a flexible linker (such as a polypeptide chain), a chemical linking
group, or any other method known in the art.
[0070] In a further aspect, the present invention provides a
composition comprising a dual-specific ligand, obtainable by a
method of the present invention, and a pharmaceutically acceptable
carrier, diluent or excipient.
[0071] Moreover, the present invention provides a method for the
treatment and/or prevention of disease using a `dual-specific
ligand` or a composition according to the present invention.
[0072] In a second configuration, the present invention provides
multispecific ligands which comprise at least two non-complementary
variable domains. For example, the ligands may comprise a pair of
V.sub.H domains or a pair of V.sub.L domains. Advantageously, the
domains are of non-camelid origin; preferably they are human
domains or comprise human framework regions (FWs) and one or more
heterologous CDRs. CDRs and framework regions are those regions of
an immunoglobulin variable domain as defined in the Kabat database
of Sequences of Proteins of Immunological Interest.
[0073] Preferred human framework regions are those encoded by
germline gene segments DP47 and DPK9. Advantageously, FW1, FW2 and
FW3 of a V.sub.H or V.sub.L domain have the sequence of FW1, FW2 or
FW3 from DP47 or DPK9. The human frameworks may optionally contain
mutations, for example up to about 5 amino acid changes or up to
about 10 amino acid changes collectively in the human frameworks
used in the ligands of the invention.
[0074] The variable domains in the multispecific ligands according
to the second configuration of the invention may be arranged in an
open or a closed conformation; that is, they may be arranged such
that the variable domains can bind their cognate ligands
independently and simultaneously, or such that only one of the
variable domains may bind its cognate ligand at any one time.
[0075] The inventors have realised that under certain structural
conditions, non-complementary variable domains (for example two
light chain variable domains or two heavy chain variable domains)
may be present in a ligand such that binding of a first epitope to
a first variable domain inhibits the binding of a second epitope to
a second variable domain, even though such non-complementary
domains do not operate together as a cognate pair.
[0076] Advantageously, the ligand comprises two or more pairs of
variable domains; that is, it comprises at least four variable
domains. Advantageously, the four variable domains comprise
frameworks of human origin.
[0077] In a preferred embodiment, the human frameworks are
identical to those of human germline sequences.
[0078] The present inventors consider that such antibodies will be
of particular use in ligand binding assays for therapeutic and
other uses.
[0079] Thus, in a first aspect of the second configuration, the
present invention provides a method for producing a multispecific
ligand comprising the steps of: [0080] a) selecting a first epitope
binding domain by its ability to bind to a first epitope, [0081] b)
selecting a second epitope binding domain by its ability to bind to
a second epitope, [0082] c) combining the epitope binding domains;
and [0083] d) selecting the closed conformation multispecific
ligand by its ability to bind to said first second epitope and said
second epitope.
[0084] In a further aspect of the second configuration, the
invention provides method for preparing a closed conformation
multi-specific ligand comprising a first epitope binding domain
having a first epitope binding specificity and a non-complementary
second epitope binding domain having a second epitope binding
specificity, wherein the first and second binding specificities
compete for epitope binding such that the closed conformation
multi-specific ligand may not bind both epitopes simultaneously,
said method comprising the steps of: [0085] a) selecting a first
epitope binding domain by its ability to bind to a first epitope,
[0086] b) selecting a second epitope binding domain by its ability
to bind to a second epitope, [0087] c) combining the epitope
binding domains such that the domains are in a closed conformation;
and [0088] d) selecting the closed conformation multispecific
ligand by its ability to bind to said first second epitope and said
second epitope, but not to both said first and second epitopes
simultaneously.
[0089] Moreover, the invention provides a closed conformation
multi-specific ligand comprising a first epitope binding domain
having a first epitope binding specificity and a non-complementary
second epitope binding domain having a second epitope binding
specificity, wherein the first and second binding specificities
compete for epitope binding such that the closed conformation
multi-specific ligand may not bind both epitopes
simultaneously.
[0090] An alternative embodiment of the above aspect of the of the
second configuration of the invention optionally comprises a
further step (b1) comprising selecting a third or further epitope
binding domain. In this way the multi-specific ligand produced,
whether of open or closed conformation, comprises more than two
epitope binding specificities. In a preferred aspect of the second
configuration of the invention, where the multi-specific ligand
comprises more than two epitope binding domains, at least two of
said domains are in a closed conformation and compete for binding;
other domains may compete for binding or may be free to associate
independently with their cognate epitope(s).
[0091] According to the present invention the term `multi-specific
ligand` refers to a ligand which possesses more than one epitope
binding specificity as herein defined.
[0092] As herein defined the term `closed conformation`
(multi-specific ligand) means that the epitope binding domains of
the ligand are attached to or associated with each other,
optionally by means of a protein skeleton, such that epitope
binding by one epitope binding domain competes with epitope binding
by another epitope binding domain. That is, cognate epitopes may be
bound by each epitope binding domain individually but not
simultaneosuly. The closed conformation of the ligand can be
achieved using methods herein described.
[0093] "Open conformation" means that the epitope binding domains
of the ligand are attached to or associated with each other,
optionally by means of a protein skeleton, such that epitope
binding by one epitope binding domain does not compete with epitope
binding by another epitope binding domain.
[0094] As referred to herein, the term `competes` means that the
binding of a first epitope to its cognate epitope binding domain is
inhibited when a second epitope is bound to its cognate epitope
binding domain. For example, binding may be inhibited sterically,
for example by physical blocking of a binding domain or by
alteration of the structure or environment of a binding domain such
that its affinity or avidity for an epitope is reduced.
[0095] In a further embodiment of the second configuration of the
invention, the epitopes may displace each other on binding. For
example, a first epitope may be present on an antigen which, on
binding to its cognate first binding domain, causes steric
hindrance of a second binding domain, or a coformational change
therein, which displaces the epitope bound to the second binding
domain.
[0096] Advantageously, binding is reduced by 25% or more,
advantageously 40%, 50%, 60%, 70%, 80%, 90% or more, and preferably
up to 100% or nearly so, such that binding is completely inhibited.
Binding of epitopes can be measured by conventional antigen binding
assays, such as ELISA, by fluorescence based techniques, including
FRET, or by techniques such as suface plasmon resonance which
measure the mass of molecules.
[0097] According to the method of the present invention,
advantageously, each epitope binding domain is of a different
epitope binding specificity.
[0098] In the context of the present invention, first and second
"epitopes" are understood to be epitopes which are not the same and
are not bound by a single monospecific ligand. They may be on
different antigens or on the same antigen, but separated by a
sufficient distance that they do not form a single entity that
could be bound by a single mono-specific V.sub.H/V.sub.L binding
pair of a conventional antibody. Experimentally, if both of the
individual variable domains in single chain antibody form (domain
antibodies or dAbs) are separately competed by a monospecific
V.sub.H/V.sub.L ligand against two epitopes then those two epitopes
are not sufficiently far apart to be considered separate epitopes
according to the present invention.
[0099] The closed conformation multispecific ligands of the
invention do not include ligands as described in WO 02/02773. Thus,
the ligands of the present invention do not comprise complementary
V.sub.H/V.sub.L pairs which bind any one or more antigens or
epitopes co-operatively. Instead, the ligands according to the
invention preferably comprise non-complementary V.sub.H-V.sub.H or
V.sub.L-V.sub.L pairs. Advantageously, each V.sub.H or V.sub.L
domain in each V.sub.H-V.sub.H or V.sub.L-V.sub.L pair has a
different epitope binding specificity, and the epitope binding
sites are so arranged that the binding of an epitope at one site
competes with the binding of an epitope at another site.
[0100] According to the present invention, advantageously, each
epitope binding domain comprises an immunoglobulin variable domain.
More advantageously, each immunoglobulin variable domain will be
either a variable light chain domain (V.sub.L) or a variable heavy
chain domain V.sub.H. In the second configuration of the present
invention, the immunoglobulin domains when present on a ligand
according to the present invention are non-complementary, that is
they do not associate to form a V.sub.H/V.sub.L antigen binding
site. Thus, multi-specific ligands as defined in the second
configuration of the invention comprise immunoglobulin domains of
the same sub-type, that is either variable light chain domains
(V.sub.L) or variable heavy chain domains (V.sub.H). Moreover,
where the ligand according to the invention is in the closed
conformation, the immunoglobulin domains may be of the camelid
V.sub.HH type.
[0101] In an alternative embodiment, the ligand(s) according to the
invention do not comprise a camelid V.sub.HH domain. More
particularly, the ligand(s) of the invention do not comprise one or
more amino acid residues that are specific to camelid V.sub.HH
domains as compared to human V.sub.H domains.
[0102] Advantageously, the single variable domains are derived from
antibodies selected for binding activity against different antigens
or epitopes. For example, the variable domains may be isolated at
least in part by human immunisation. Alternative methods are known
in the art, including isolation from human antibody libraries and
synthesis of artificial antibody genes.
[0103] The variable domains advantageously bind superantigens, such
as protein A or protein L. Binding to superantigens is a property
of correctly folded antibody variable domains, and allows such
domains to be isolated from, for example, libraries of recombinant
or mutant domains.
[0104] Epitope binding domains according to the present invention
comprise a protein scaffold and epitope interaction sites (which
are advantageously on the surface of the protein scaffold).
[0105] Epitope binding domains may also be based on protein
scaffolds or skeletons other than immunoglobulin domains. For
example natural bacterial receptors such as SpA have been used as
scaffolds for the grafting of CDRs to generate ligands which bind
specifically to one or more epitopes. Details of this procedure are
described in U.S. Pat. No. 5,831,012. Other suitable scaffolds
include those based on fibronectin and affibodies. Details of
suitable procedures are described in WO 98/58965. Other suitable
scaffolds include lipocallin and CTLA4, as described in van den
Beuken et al., J. Mol. Biol. (2001) 310, 591-601, and scaffolds
such as those described in WO0069907 (Medical Research Council),
which are based for example on the ring structure of bacterial
GroEL or other chaperone polypeptides.
[0106] Protein scaffolds may be combined; for example, CDRs may be
grafted on to a CTLA4 scaffold and used together with
immunoglobulin V.sub.H or V.sub.L domains to form a multivalent
ligand. Likewise, fibronectin, lipocallin and other scaffolds may
be combined.
[0107] It will be appreciated by one skilled in the art that the
epitope binding domains of a closed conformation multispecific
ligand produced according to the method of the present invention
may be on the same polypeptide chain, or alternatively, on
different polypeptide chains. In the case that the variable regions
are on different polypeptide chains, then they may be linked via a
linker, advantageously a flexible linker (such as a polypeptide
chain), a chemical linking group, or any other method known in the
art.
[0108] The first and the second epitope binding domains may be
associated either covalently or non-covalently. In the case that
the domains are covalently associated, then the association may be
mediated for example by disulphide bonds.
[0109] In the second configuation of the invention, the first and
the second epitopes are preferably different. They may be, or be
part of, polypeptides, proteins or nucleic acids, which may be
naturally occurring or synthetic. In this respect, the ligand of
the invention may bind an epiotpe or antigen and act as an
antagonist or agonist (eg, EPO receptor agonist). The epitope
binding domains of the ligand in one embodiment have the same
epitope specificity, and may for example simultaneously bind their
epitope when multiple copies of the epitope are present on the same
antigen. In another embodiment, these epitopes are provided on
different antigens such that the ligand can bind the epitopes and
bridge the antigens. One skilled in the art will appreciate that
the choice of epitopes and antigens is large and varied. They may
be for instance human or animal proteins, cytokines, cytokine
receptors, enzymes co-factors for enzymes or DNA binding proteins.
Suitable cytokines and growth factors include but are not limited
to: ApoE, Apo-SAA, BDNF, Cardiotrophin-1, EGF, EGF receptor,
ENA-78, Eotaxin, Eotaxin-2, Exodus-2, EpoR, FGF-acidic, FGF-basic,
fibroblast growth factor-10, FLT3 ligand, Fractalkine (CX3C), GDNF,
G-CSF, GM-CSF, GF-.beta.1, insulin, IFN-.gamma., IGF-I, IGF-II,
IL-1.alpha., IL-1.beta., IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8
(72 a.a.), IL-8 (77 a.a.), IL-9, IL-10, IL-11, IL-12, IL-13, IL-15,
IL-16, IL-17, IL-18 (IGIF), Inhibin .alpha., Inhibin .beta., IP-10,
keratinocyte growth factor-2 (KGF-2), KGF, Leptin, LIF,
Lymphotactin, Mullerian inhibitory substance, monocyte colony
inhibitory factor, monocyte attractant protein, M-CSF, MDC (67
a.a.), MDC (69 a.a.), MCP-1 (MCAF), MCP-2, MCP-3, MCP-4, MDC (67
a.a.), MDC (69 a.a.), MIG, MIP-1.alpha., MIP-1.beta., MIP-3.alpha.,
MIP-3.beta., MIP-4, myeloid progenitor inhibitor factor-1 (MPIF-1),
NAP-2, Neurturin, Nerve growth factor, .beta.-NGF, NT-3, NT-4,
Oncostatin M, PDGF-AA, PDGF-AB, PDGF-BB, PF-4, RANTES, SDF1.alpha.,
SDF1.beta., SCF, SCGF, stem cell factor (SCF), TARC, TGF-.alpha.,
TGF-.beta., TGF-.beta.2, TGF-.beta.3, tumour necrosis factor (TNF),
TNF-.alpha., TNF-.beta., TNF receptor I, TNF receptor II, TNIL-1,
TPO, VEGF, VEGF receptor 1, VEGF receptor 2, VEGF receptor 3,
GCP-2, GRO/MGSA, GRO-.beta., GRO-.gamma., HCC1, 1-309, HER 1, HER
2, HER 3, HER 4, TACE recognition site, TNF BP-I and TNF BP-II, as
well as any target disclosed in Annex 2 or Annex 3 hereto, whether
in combination as set forth in the Annexes, in a different
combination or individually. Cytokine receptors include receptors
for the foregoing cytokines, e.g. IL-1 R1; IL-6R; IL-10R; IL-18R,
as well as receptors for cytokines set forth in Annex 2 or Annex 3
and also receptors disclosed in Annex 2 and 3. It will be
appreciated that this list is by no means exhaustive. Where the
multispecific ligand binds to two epitopes (on the same or
different antigens), the antigen(s) may be selected from this
list.
[0110] Advantageously, dual specific ligands may be used to target
cytokines and other molecules which cooperate synergistically in
therapeutic situations in the body of an organism. The invention
therefore provides a method for synergising the activity of two or
more cytokines, comprising administering a dual specific ligand
capable of binding to said two or more cytokines. In this aspect of
the invention, the dual specific ligand may be any dual specific
ligand, including a ligand composed of complementary and/or
non-complementary domains, a ligand in an open conformation, and a
ligand in a closed conformation. For example, this aspect of the
invention relates to combinations of V.sub.H domains and V.sub.L
domains, V.sub.H domains only and V.sub.L domains only.
[0111] Synergy in a therapeutic context may be achieved in a number
of ways. For example, target combinations may be therapeutically
active only if both targets are targeted by the ligand, whereas
targeting one target alone is not therapeutically effective. In
another embodiment, one target alone may provide some low or
minimal therapeutic effect, but together with a second target the
combination provides a synergistic increase in therapeutic
effect.
[0112] Preferably, the cytokines bound by the dual specific ligands
of this aspect of the invention are slected from the list shown in
Annex 2.
[0113] Moreover, dual specific ligands may be used in oncology
applications, where one specificity targets CD89, which is
expressed by cytotoxic cells, and the other is tumour specific.
Examples of tumour antigens which may be targetted are given in
Annex 3.
[0114] In one embodiment of the second configuration of the
invention, the variable domains are derived from an antibody
directed against the first and/or second antigen or epitope. In a
preferred embodiment the variable domains are derived from a
repertoire of single variable antibody domains. In one example, the
repertoire is a repertoire that is not created in an animal or a
synthetic repertoire. In another example, the single variable
domains are not isolated (at least in part) by animal immunisation.
Thus, the single domains can be isolated from a naive library.
[0115] The second configuration of the invention, in another
aspect, provides a multi-specific ligand comprising a first epitope
binding domain having a first epitope binding specificity and a
non-complementary second epitope binding domain having a second
epitope binding specificity. The first and second binding
specificities may be the same or different.
[0116] In a further aspect, the present invention provides a closed
conformation multi-specific ligand comprising a first epitope
binding domain having a first epitope binding specificity and a
non-complementary second epitope binding domain having a second
epitope binding specificity wherein the first and second binding
specificities are capable of competing for epitope binding such
that the closed conformation multi-specific ligand cannot bind both
epitopes simultaneously.
[0117] In a still further aspect, the invention provides open
conformation ligands comprising non-complementary binding domains,
wherein the deomains are specific for a different epitope on the
same target. Such ligands bind to targets with increased avidity.
Similarly, the invention provides multivalent ligands comprising
non-complementary binding domains specific for the same epitope and
directed to targets which comprise multiple copies of said epitope,
such as IL-5, PDGF-AA, PDGF-BB, TGF beta, TGF beta2, TGF beta3 and
TNF.alpha., for eample human TNF Receptor 1 and human
TNF.alpha..
[0118] In a similar aspect, ligands according to the invention can
be configured to bind individual epitopes with low affinity, such
that binding to individual epitopes is not therapeutically
significant; but the increased avidity resulting from binding to
two epitopes provides a theapeutic benefit. In a perticular
example, epitopes may be targetted which are present individually
on normal cell types, but present together only on abnormal or
diseased cells, such as tumour cells. In such a situaton, only the
abnormal or diseased cells are effectively targetted by the
bispecific ligands according to the invention.
[0119] Ligand specific for multiple copies of the same epitope, or
adjacent epitopes, on the same target (known as chelating dAbs) may
also be trimeric or polymeric (tertrameric or more) ligands
comprising three, four or more non-complementary binding domains.
For example, ligands may be constructed comprising three or four
V.sub.H domains or V.sub.L domains.
[0120] Moreover, ligands are provided which bind to multisubunit
targets, wherein each binding domain is specific for a subunit of
said target. The ligand may be dimeric, trimeric or polymeric.
[0121] Preferably, the multi-specific ligands according to the
above aspects of the invention are obtainable by the method of the
first aspect of the invention.
[0122] According to the above aspect of the second configuration of
the invention, advantageously the first epitope binding domain and
the second epitope binding domains are non-complementary
immunoglobulin variable domains, as herein defined. That is either
V.sub.H-V.sub.H or V.sub.L-V.sub.L variable domains.
[0123] Chelating dAbs in particular may be prepared according to a
preferred aspect of the invention, namely the use of anchor dAbs,
in which a library of dimeric, trimeric or multimeric dAbs is
constructed using a vector which comprises a constant dAb upstream
or downstream of a linker sequence, with a repertoire of second,
third and further dAbs being inserted on the other side of the
linker. For example, the anchor or guiding dAb may be TAR1-5
(V.sub..kappa.), TAR1-27(V.sub..kappa.), TAR2h-5(V.sub.H) or
TAR2h-6(V.sub..kappa.).
[0124] In alternative methodologies, the use of linkers may be
avoided, for example by the use of non-covalent bonding or naturall
affinity between binding domains such as V.sub.H and V.sub..kappa..
The invention accordingly provides a method for preparing a
chelating multimeric ligand comprising the steps of: [0125] (a)
providing a vector comprising a nucleic acid sequence encoding a
single binding domain specific for a first epitope on a target;
[0126] (b) providing a vector encoding a repertoire comprising
second binding domains specific for a second epitope on said
target, which epitope can be the same or different to the first
epitope, said second epitope being adjacent to said first epitope;
and [0127] (c) expressing said first and second binding domains;
and [0128] (d) isolating those combinations of first and second
binding domains which combine together to produce a target-binding
dimer.
[0129] The first and second epitopes are adjacent such that a
multimeric ligand is capable of binding to both epitopes
simultaneously. This provides the ligand with the advantages of
increased avidity if binding. Where the epitopes are the same, the
increased avidity is obtained by the presence of multiple copies of
the epitope on the target, allowing at least two copies to be
simultaneously bound in order to obtain the increased avidity
effect.
[0130] The binding domains may be associated by several methods, as
well as the use of linkers. For example, the binding domains may
comprise cys residues, avidin and streptavidin groups or other
means for non-covalent attachment post-synthesis; those
combinations which bind to the target efficiently will be isolated.
Alternatively, a linker may be present between the first and second
binding domains, which are expressed as a single polypeptide from a
single vector, which comprises the first binding domain, the linker
and a repertoire of second binding domains, for instance as
described above.
[0131] In a preferred aspect, the first and second binding domains
associate naturally when bound to antigen; for example, V.sub.H and
V.sub..kappa. domains, when bound to adjacent epitopes, will
naturally associate in a three-way interaction to form a stable
dimer. Such associated proteins can be isolated in a target binding
assay. An advantage of this procedure is that only binding domains
which bind to closely adjacent epitopes, in the correct
conformation, will associate and thus be isolated as a result of
their increased avidity for the target.
[0132] In an alternative embodiment of the above aspect of the
second configuration of the invention, at least one epitope binding
domain comprises a non-immunoglobulin `protein scaffold` or
`protein skeleton` as herein defined. Suitable non-immunoglobulin
protein scaffolds include but are not limited to any of those
selected from the group consisting of: SpA, fibronectin, GroEL and
other chaperones, lipocallin, CCTLA4 and affibodies, as set forth
above.
[0133] According to the above aspect of the second configuration of
the invention, advantageously, the epitope binding domains are
attached to a `protein skeleton`. Advantageously, a protein
skeleton according to the invention is an immunoglobulin
skeleton.
[0134] According to the present invention, the term `immunoglobulin
skeleton` refers to a protein which comprises at least one
immunoglobulin fold and which acts as a nucleus for one or more
epitope binding domains, as defined herein.
[0135] Preferred immunoglobulin skeletons as herein defined
includes any one or more of those selected from the following: an
immunoglobulin molecule comprising at least (i) the CL (kappa or
lambda subclass) domain of an antibody; or (ii) the CH1 domain of
an antibody heavy chain; an immunoglobulin molecule comprising the
CH1 and CH2 domains of an antibody heavy chain; an immunoglobulin
molecule comprising the CH1, CH2 and CH3 domains of an antibody
heavy chain; or any of the subset (ii) in conjunction with the CL
(kappa or lambda subclass) domain of an antibody. A hinge region
domain may also be included. Such combinations of domains may, for
example, mimic natural antibodies, such as IgG or IgM, or fragments
thereof, such as Fv, scFv, Fab or F(ab').sub.2 molecules. Those
skilled in the art will be aware that this list is not intended to
be exhaustive.
[0136] Linking of the skeleton to the epitope binding domains, as
herein defined may be achieved at the polypeptide level, that is
after expression of the nucleic acid encoding the skeleton and/or
the epitope binding domains. Alternatively, the linking step may be
performed at the nucleic acid level. Methods of linking a protein
skeleton according to the present invention, to the one or more
epitope binding domains include the use of protein chemistry and/or
molecular biology techniques which will be familiar to those
skilled in the art and are described herein.
[0137] Advantageously, the closed conformation multispecific ligand
may comprise a first domain capable of binding a target molecule,
and a second domain capable of binding a molecule or group which
extends the half-life of the ligand. For example, the molecule or
group may be a bulky agent, such as HSA or a cell matrix protein.
As used herein, the phrase "molecule or group which extends the
half-life of a ligand" refers to a molecule or chemical group
which, when bound by a dual-specific ligand as described herein
increases the in vivo half-life of such dual specific ligand when
administered to an animal, relative to a ligand that does not bind
that molecule or group. Examples of molecules or groups that extend
the half-life of a ligand are described hereinbelow. In a preferred
embodiment, the closed conformation multispecific ligand may be
capable of binding the target molecule only on displacement of the
half-life enhancing molecule or group. Thus, for example, a closed
conformation multispecific ligand is maintained in circulation in
the bloodstream of a subject by a bulky molecule such as HSA. When
a target molecule is encountered, competition between the binding
domains of the closed conformation multispecific ligand results in
displacement of the HSA and binding of the target.
[0138] Ligands according to any aspect of the present invention, as
well as dAb monomers useful in constructing such ligands, may
advantageously dissociate from their cognate target(s) with a
K.sub.d of 300 nM to 5 pM (ie, 3.times.10.sup.-7 to
5.times.10.sup.-12M), preferably 50 nM to 20 pM, or 5 nM to 200 pM
or 1 nM to 100 pM, 1.times.10.sup.-7 M or less, 1.times.10.sup.-8 M
or less, 1.times.10.sup.-9 M or less, 1.times.10.sup.-10 M or less,
1.times.10.sup.-11 M or less; and/or a K.sub.off rate constant of
5.times.10.sup.-1 to 1.times.10.sup.-7 S.sup.-1, preferably
1.times.10.sup.-2 to 1.times.10.sup.-6 S.sup.-1, or
5.times.10.sup.-3 to 1.times.10.sup.-5 S.sup.-1, or
5.times.10.sup.-1 S.sup.-1 or less, or 1.times.10.sup.-2 S.sup.-1
or less, or 1.times.10.sup.-3 S.sup.-1 or less, or
1.times.10.sup.-4 S.sup.-1 or less, or 1.times.10.sup.-5 S.sup.-1
or less, or 1.times.10.sup.-6 S.sup.-1 or less as determined by
surface plasmon resonance. The K.sub.d rate constand is defined as
K.sub.off/K.sub.on.
[0139] In particular the invention provides an anti-TNF.alpha. dAb
monomer (or dual specific ligand comprising such a dAb), homodimer,
heterodimer or homotrimer ligand, wherein each dAb binds
TNF.alpha.. The ligand binds to TNF.alpha. with a K.sub.d of 300 nM
to 5 pM (ie, 3.times.10.sup.-7 to 5.times.10.sup.-12M), preferably
50 nM to 20 pM, more preferably 5 nM to 200 pM and most preferably
1 nM to 100 pM; expressed in an alternative manner, the K.sub.d is
1.times.10.sup.-7 M or less, preferably 1.times.10.sup.-8 M or
less, more preferably 1.times.10.sup.-9 M or less, advantageously
1.times.10.sup.-10 M or less and most preferably 1.times.10.sup.-11
M or less; and/or a K.sub.off rate constant of 5.times.10.sup.-1 to
1.times.10.sup.-7 S.sup.-1, preferably 1.times.10.sup.-2 to
1.times.10.sup.-6 S.sup.-1, more preferably 5.times.10.sup.-3 to
1.times.10.sup.-5 S.sup.-1, for example 5.times.10.sup.-1 S.sup.-1
or less, preferably 1.times.10.sup.-2 S.sup.-1 or less, more
preferably 1.times.10.sup.-3 S.sup.-1 or less, advantageously
1.times.10.sup.-4 S.sup.-1 or less, further advantageously
1.times.10.sup.-5 S.sup.-1 or less, and most preferably
1.times.10.sup.-6 S.sup.-1 or less, as determined by surface
plasmon resonance.
[0140] Preferably, the ligand neutralises TNF.alpha. in a standard
L929 assay with an ND50 of 500 nM to 50 pM, preferably or 100 nM to
50 pM, advantageously 10 nM to 100 pM, more preferably 1 nM to 100
pM; for example 50 nM or less, preferably 5 nM or less,
advantageously 500 pM or less, more preferably 200 pM or less and
most preferably 100 pM or less.
[0141] Preferably, the ligand inhibits binding of TNF alpha to TNF
alpha Receptor I (p55 receptor) with an IC50 of 500 nM to 50 pM,
preferably 100 nM to 50 pM, more preferably 10 nM to 100 pM,
advantageously 1 nM to 100 pM; for example 50 nM or less,
preferably 5 nM or less, more preferably 500 pM or less,
advantageously 200 pM or less, and most preferably 100 pM or less.
Preferably, the TNF.alpha. is Human TNF.alpha..
[0142] Furthermore, the invention provides a an anti-TNF Receptor I
dAb monomer, or dual specific ligand comprising such a dAb, that
binds to TNF Receptor I with a K.sub.d of 300 nM to 5 pM (ie,
3.times.10.sup.-7 to 5.times.10.sup.-12M), preferably 50 nM to 20
pM, more preferably 5 nM to 200 pM and most preferably 1 nM to 100
pM, for example 1.times.10.sup.-7 M or less, preferably
1.times.10.sup.-8 M or less, more preferably 1.times.10.sup.-9 M or
less, advantageously 1.times.10.sup.-10 M or less and most
preferably 1.times.10.sup.-11 M or less; and/or a K.sub.off rate
constant of 5.times.10.sup.-1 to 1.times.10.sup.-7 S.sup.-1,
preferably 1.times.10.sup.-2 to 1.times.10.sup.-6 S.sup.-1, more
preferably 5.times.10.sup.-3 to 1.times.10.sup.-5 S.sup.-1, for
example 5.times.10.sup.-1 S.sup.-1 or less, preferably
1.times.10.sup.-2 S.sup.-1 or less, advantageously
1.times.10.sup.-3 S.sup.-1 or less, more preferably
1.times.10.sup.-4 S.sup.-1 or less, still more preferably
1.times.10.sup.-5 S.sup.-1 or less, and most preferably
1.times.10.sup.-6 S.sup.-1 or less as determined by surface plasmon
resonance.
[0143] Preferably, the dAb monomeror ligand neutralises TNF.alpha.
in a standard assay (eg, the L929 or HeLa assays described herein)
with an ND50 of 500 nM to 50 pM, preferably 100 nM to 50 pM, more
preferably 10 nM to 100 pM, advantageously 1 nM to 100 pM; for
example 50 nM or less, preferably 5 nM or less, more preferably 500
pM or less, advantageously 200 pM or less, and most preferably 100
pM or less.
[0144] Preferably, the dAb monomer or ligand inhibits binding of
TNF alpha to TNF alpha Receptor I (p55 receptor) with an IC50 of
500 nM to 50 pM, preferably 100 nM to 50 pM, more preferably 10 nM
to 100 pM, advantageously 1 nM to 100 pM; for example 50 nM or
less, preferably 5 nM or less, more preferably 500 pM or less,
advantageously 200 pM or less, and most preferably 100 pM or less.
Preferably, the TNF Receptor I target is Human TNF.alpha..
[0145] Furthermore, the invention provides a dAb monomer(or dual
specific ligand comprising such a dAb) that binds to serum albumin
(SA) with a K.sub.d of 1 nM to 500 .mu.M (ie, .times.10.sup.-9 to
5.times.10.sup.-4), preferably 100 nM to 10 .mu.M. Preferably, for
a dual specific ligand comprising a first anti-SA dAb and a second
dAb to another target, the affinity (eg K.sub.d and/or K.sub.off as
measured by surface plasmon resonance, eg using BiaCore) of the
second dAb for its target is from 1 to 100000 times (preferably 100
to 100000, more preferably 1000 to 100000, or 10000 to 100000
times) the affinity of the first dAb for SA. For example, the first
dAb binds SA with an affinity of approximately 10 .mu.M, while the
second dAb binds its target with an affinity of 100 pM. Preferably,
the serum albumin is human serum albumin (HSA).
[0146] In one embodiment, the first dAb (or a dAb monomer) binds SA
(eg, HSA) with a K.sub.d of approximately 50, preferably 70, and
more preferably 100, 150 or 200 nM.
[0147] The invention moreover provides dimers, trimers and polymers
of the aforementioned dAb monomers, in accordance with the
foregoing aspect of the present invention.
[0148] Ligands according to the invention, including dAb monomers,
dimers and trimers, can be linked to an antibody Fc region,
comprising one or both of C.sub.H2 and C.sub.H3 domains, and
optionally a hinge region. For example, vectors encoding ligands
linked as a single nucleotide sequence to an Fc region may be used
to prepare such polypeptides.
[0149] In a further aspect of the second configuration of the
invention, the present invention provides one or more nucleic acid
molecules encoding at least a multispecific ligand as herein
defined. In one embodiment, the ligand is a closed conformation
ligand. In another embodiment, it is an open conformation ligand.
The multispecific ligand may be encoded on a single nucleic acid
molecule; alternatively, each epitope binding domain may be encoded
by a separate nucleic acid molecule. Where the ligand is encoded by
a single nucleic acid molecule, the domains may be expressed as a
fusion polypeptide, or may be separately expressed and subsequently
linked together, for example using chemical linking agents. Ligands
expressed from separate nucleic acids will be linked together by
appropriate means.
[0150] The nucleic acid may further encode a signal sequence for
export of the polypeptides from a host cell upon expression and may
be fused with a surface component of a filamentous bacteriophage
particle (or other component of a selection display system) upon
expression. Leader sequences, which may be used in bacterial
expresion and/or phage or phagemid display, include pelB, stII,
ompA, phoA, bla and pelA.
[0151] In a further aspect of the second configuration of the
invention the present invention provides a vector comprising
nucleic acid according to the present invention.
[0152] In a yet further aspect, the present invention provides a
host cell transfected with a vector according to the present
invention.
[0153] Expression from such a vector may be configured to produce,
for example on the surface of a bacteriophage particle, epitope
binding domains for selection. This allows selection of displayed
domains and thus selection of `multispecific ligands` using the
method of the present invention.
[0154] In a preferred embodiment of the second configuration of the
invention, the epitope binding domains are immunoglobulin variable
regions and are selected from single domain V gene repertoires.
Generally the repertoire of single antibody domains is displayed on
the surface of filamentous bacteriophage. In a preferred embodiment
each single antibody domain is selected by binding of a phage
repertoire to antigen.
[0155] The present invention further provides a kit comprising at
least a multispecific ligand according to the present invention,
which may be an open conformation or closed conformation ligand.
Kits according to the invention may be, for example, diagnostic
kits, therapeutic kits, kits for the detection of chemical or
biological species, and the like.
[0156] In a further aspect still of the second configuration of the
invention, the present invention provides a homogenous immunoassay
using a ligand according to the present invention.
[0157] In a further aspect still of the second configuration of the
invention, the present invention provides a composition comprising
a closed conformation multispecific ligand, obtainable by a method
of the present invention, and a pharmaceutically acceptable
carrier, diluent or excipient.
[0158] Moreover, the present invention provides a method for the
treatment of disease using a `closed conformation multispecific
ligand` or a composition according to the present invention.
[0159] In a preferred embodiment of the invention the disease is
cancer or an inflammatory disease, eg rheumatoid arthritis, asthma
or Crohn's disease.
[0160] In a further aspect of the second configuration of the
invention, the present invention provides a method for the
diagnosis, including diagnosis of disease using a closed
conformation multispecific ligand, or a composition according to
the present invention. Thus in general the binding of an analyte to
a closed conformation multispecific ligand may be exploited to
displace an agent, which leads to the generation of a signal on
displacement. For example, binding of analyte (second antigen)
could displace an enzyme (first antigen) bound to the antibody
providing the basis for an immunoassay, especially if the enzyme
were held to the antibody through its active site.
[0161] Thus in a final aspect of the second configuration, the
present invention provides a method for detecting the presence of a
target molecule, comprising:
[0162] (a) providing a closed conformation multispecific ligand
bound to an agent, said ligand being specific for the target
molecule and the agent, wherein the agent which is bound by the
ligand leads to the generation of a detectable signal on
displacement from the ligand;
[0163] (b) exposing the closed conformation multispecific ligand to
the target molecule; and
[0164] (c) detecting the signal generated as a result of the
displacement of the agent.
[0165] According to the above aspect of the second configuration of
the invention, advantageously, the agent is an enzyme, which is
inactive when bound by the closed conformation multi-specific
ligand. Alternatively, the agent may be any one or more selected
from the group consisting of the following: the substrate for an
enzyme, and a fluorescent, luminescent or chromogenic molecule
which is inactive or quenched when bound by the ligand.
[0166] Sequences similar or homologous (e.g., at least about 70%
sequence identity) to the sequences disclosed herein are also part
of the invention. In some embodiments, the sequence identity at the
amino acid level can be about 80%, 85%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99% or higher. At the nucleic acid level, the
sequence identity can be about 70%, 75%, 80%, 85%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99% or higher. Alternatively,
substantial identity exists when the nucleic acid segments will
hybridize under selective hybridization conditions (e.g., very high
stringency hybridization conditions), to the complement of the
strand. The nucleic acids may be present in whole cells, in a cell
lysate, or in a partially purified or substantially pure form.
[0167] Calculations of "homology" or "sequence identity" or
"similarity" between two sequences (the terms are used
interchangeably herein) are performed as follows. The sequences are
aligned for optimal comparison purposes (e.g., gaps can be
introduced in one or both of a first and a second amino acid or
nucleic acid sequence for optimal alignment and non-homologous
sequences can be disregarded for comparison purposes). In a
preferred embodiment, the length of a reference sequence aligned
for comparison purposes is at least 30%, preferably at least 40%,
more preferably at least 50%, even more preferably at least 60%,
and even more preferably at least 70%, 80%, 90%, 100% of the length
of the reference sequence. The amino acid residues or nucleotides
at corresponding amino acid positions or nucleotide positions are
then compared. When a position in the first sequence is occupied by
the same amino acid residue or nucleotide as the corresponding
position in the second sequence, then the molecules are identical
at that position (as used herein amino acid or nucleic acid
"homology" is equivalent to amino acid or nucleic acid "identity").
The percent identity between the two sequences is a function of the
number of identical positions shared by the sequences, taking into
account the number of gaps, and the length of each gap, which need
to be introduced for optimal alignment of the two sequences.
[0168] Advantageously, the BLAST algorithm (version 2.0) is
employed for sequence alignment, with parameters set to default
values. The BLAST algorithm is described in detail at the world
wide web site ("www") of the National Center for Biotechnology
Information (".ncbi") of the National Institutes of Health ("nih")
of the U.S. government (".gov"), in the "/Blast/" directory, in the
"blast_help.html" file. The search parameters are defined as
follows, and are advantageously set to the defined default
parameters.
[0169] BLAST (Basic Local Alignment Search Tool) is the heuristic
search algorithm employed by the programs blastp, blastn, blastx,
tblastn, and tblastx; these programs ascribe significance to their
findings using the statistical methods of Karlin and Altschul,
1990, Proc. Natl. Acad. Sci. USA 87(6):2264-8 (see the
"blast_help.html" file, as described above) with a few
enhancements. The BLAST programs were tailored for sequence
similarity searching, for example to identify homologues to a query
sequence. The programs are not generally useful for motif-style
searching. For a discussion of basic issues in similarity searching
of sequence databases, see Altschul et al. (1994).
[0170] The five BLAST programs available at the National Center for
Biotechnology Information web site perform the following tasks:
[0171] "blastp" compares an amino acid query sequence against a
protein sequence database;
[0172] "blastn" compares a nucleotide query sequence against a
nucleotide sequence database;
[0173] "blastx" compares the six-frame conceptual translation
products of a nucleotide query sequence (both strands) against a
protein sequence database;
[0174] "tblastn" compares a protein query sequence against a
nucleotide sequence database dynamically translated in all six
reading frames (both strands).
[0175] "tblastx" compares the six-frame translations of a
nucleotide query sequence against the six-frame translations of a
nucleotide sequence database.
[0176] BLAST uses the following search parameters:
[0177] HISTOGRAM Display a histogram of scores for each search;
default is yes. (See parameter H in the BLAST Manual).
[0178] DESCRIPTIONS Restricts the number of short descriptions of
matching sequences reported to the number specified; default limit
is 100 descriptions. (See parameter V in the manual page). See also
EXPECT and CUTOFF.
[0179] ALIGNMENTS Restricts database sequences to the number
specified for which high-scoring segment pairs (HSPs) are reported;
the default limit is 50. If more database sequences than this
happen to satisfy the statistical significance threshold for
reporting (see EXPECT and CUTOFF below), only the matches ascribed
the greatest statistical significance are reported. (See parameter
B in the BLAST Manual).
[0180] EXPECT The statistical significance threshold for reporting
matches against database sequences; the default value is 10, such
that 10 matches are expected to be found merely by chance,
according to the stochastic model of Karlin and Altschul (1990). If
the statistical significance ascribed to a match is greater than
the EXPECT threshold, the match will not be reported. Lower EXPECT
thresholds are more stringent, leading to fewer chance matches
being reported. Fractional values are acceptable. (See parameter E
in the BLAST Manual).
[0181] CUTOFF Cutoff score for reporting high-scoring segment
pairs. The default value is calculated from the EXPECT value (see
above). HSPs are reported for a database sequence only if the
statistical significance ascribed to them is at least as high as
would be ascribed to a lone HSP having a score equal to the CUTOFF
value. Higher CUTOFF values are more stringent, leading to fewer
chance matches being reported. (See parameter S in the BLAST
Manual). Typically, significance thresholds can be more intuitively
managed using EXPECT.
[0182] MATRIX Specify an alternate scoring matrix for BLASTP,
BLASTX, TBLASTN and TBLASTX. The default matrix is BLOSUM62
(Henikoff & Henikoff, 1992, Proc. Natl. Aacad. Sci. USA
89(22):10915-9). The valid alternative choices include: PAM40,
PAM120, PAM250 and IDENTITY. No alternate scoring matrices are
available for BLASTN; specifying the MATRIX directive in BLASTN
requests returns an error response.
[0183] STRAND Restrict a TBLASTN search to just the top or bottom
strand of the database sequences; or restrict a BLASTN, BLASTX or
TBLASTX search to just reading frames on the top or bottom strand
of the query sequence.
[0184] FILTER Mask off segments of the query sequence that have low
compositional complexity, as determined by the SEG program of
Wootton & Federhen (1993) Computers and Chemistry 17:149-163,
or segments consisting of short-periodicity internal repeats, as
determined by the XNU program of Claverie & States, 1993,
Computers and Chemistry 17:191-201, or, for BLASTN, by the DUST
program of Tatusov and Lipman (see the world wide web site of the
NCBI). Filtering can eliminate statistically significant but
biologically uninteresting reports from the blast output (e.g.,
hits against common acidic-, basic- or proline-rich regions),
leaving the more biologically interesting regions of the query
sequence available for specific matching against database
sequences.
[0185] Low complexity sequence found by a filter program is
substituted using the letter "N" in nucleotide sequence (e.g., "N"
repeated 13 times) and the letter "X" in protein sequences (e.g.,
"X" repeated 9 times).
[0186] Filtering is only applied to the query sequence (or its
translation products), not to database sequences. Default filtering
is DUST for BLASTN, SEG for other programs.
[0187] It is not unusual for nothing at all to be masked by SEG,
XNU, or both, when applied to sequences in SWISS-PROT, so filtering
should not be expected to always yield an effect.
[0188] Furthermore, in some cases, sequences are masked in their
entirety, indicating that the statistical significance of any
matches reported against the unfiltered query sequence should be
suspect.
[0189] NCBI-gi Causes NCBI gi identifiers to be shown in the
output, in addition to the accession and/or locus name.
[0190] Most preferably, sequence comparisons are conducted using
the simple BLAST search algorithm provided at the NCBI world wide
web site described above, in the "/BLAST" directory.
BRIEF DESCRIPTION OF THE DRAWINGS
[0191] FIG. 1 shows the diversification of V.sub.H/HSA at positions
H50, H52, H52a, H53, H55, H56, H58, H95, H96, H97, H98 (DVT or NNK
encoded respectively) which are in the antigen binding site of
V.sub.H HSA. (SEQ ID NO:1, nucleotide sequence; SEQ ID NO:2, amino
acid sequence.) The sequence of V.sub.K is diversified at positions
L50, L53.
[0192] FIG. 2 is a schematic showing the structure of the phagemid
pIT1/pIT2 used to prepare single chain Fv (scFv) libraries, and
shows the nucleotide sequence of the phagemid across the expression
control and cloning regions (SEQ ID NO:3) and the encoded amino
acid sequence (SEQ ID NO:4). The phagemid was used to prepare
[0193] Library 1: Germline V.sub.K/DVT V.sub.H, [0194] Library 2:
Germline V.sub.K/NNK V.sub.H, [0195] Library 3: Germline
V.sub.H/DVT V.sub.K, and [0196] Library 4: Germline V.sub.H/NNK
V.sub.K in phage display/ScFv format. These libraries were
pre-selected for binding to generic ligands protein A and protein L
so that the majority of the clones and selected libraries are
functional. Libraries were selected on HSA (first round) and
.beta.-gal (second round) or HSA .beta.-gal selection or on
.beta.-gal (first round) and HSA (second round) .beta.-gal HSA
selection. Soluble scFv from these clones of PCR are amplified in
the sequence. One clone encoding a dual specific antibody K8 was
chosen for further work.
[0197] FIG. 3 shows an alignment of V.sub.H chains (V.sub.H dummy
(SEQ ID NO:5), K8 (SEQ ID NO:6), VH2 (SEQ ID NO:7), VH4 (SEQ ID
NO:8), VHC11 (SEQ ID NO:9), VHA10sd (SEQ ID NO:10), VHA1sd (SEQ ID
NO:11), VHA5sd (SEQ ID NO:12), VHC5sd (SEQ ID NO:13), VHC11sd (SEQ
ID NO:14) and V.sub..kappa. chains (Vk dummy (SEQ ID NO:15), K8
(SEQ ID NO:16), E5sc (SEQ ID NO:17), C3 (SEQ ID NO:18)).
[0198] FIG. 4 shows the characterisation of the binding properties
of the K8 antibody, the binding properties of the K8 antibody
characterised by monoclonal phage ELISA, the dual specific K8
antibody was found to bind HSA and .beta.-gal and displayed on the
surface of the phage with absorbant signals greater than 1.0. No
cross reactivity with other proteins was detected.
[0199] FIG. 5 shows soluble scFv ELISA performed using known
concentrations of the K8 antibody fragment. A 96-well plate was
coated with 100 .mu.g of HSA, BSA and .beta.-gal at 10 .mu.g/ml and
100 .mu.g/ml of Protein A at 1 .mu.g/ml concentration. 50 .mu.g of
the serial dilutions of the K8 scFv was applied and the bound
antibody fragments were detected with Protein L-HRP. ELISA results
confirm the dual specific nature of the K8 antibody.
[0200] FIG. 6 shows the binding characteristics of the clone
K8V.sub.K/dummy V.sub.H analysed using soluble scFv ELISA.
Production of the soluble scFv fragments was induced by IPTG as
described by Harrison et al, Methods Enzymol. 1996;267:83-109 and
the supernatant containing scFv assayed directly. Soluble scFv
ELISA is performed as described in example 1 and the bound scFvs
were detected with Protein L-HRP. The ELISA results revealed that
this clone was still able to bind .beta.-gal, whereas binding BSA
was abolished.
[0201] FIG. 7 shows the sequence (SEQ ID NO:2 and SEQ ID NO:3) of
variable domain vectors 1 and 2.
[0202] FIG. 8 is a map of the C.sub.H vector used to construct a
V.sub.H1/V.sub.H2 multipsecific ligand.
[0203] FIG. 9 is a map of the V.sub..kappa. vector used to
construct a V.sub..kappa.1/V.sub..kappa.2 multispecific ligand.
[0204] FIG. 10 TNF receptor assay comparing TAR1-5 dimer 4,
TAR1-5-19 dimer 4 and TAR1-5-19 monomer.
[0205] FIG. 11 TNF receptor assay comparing TAR1-5 dimers 1-6. All
dimers have been FPLC purified and the results for the optimal
dimeric species are shown.
[0206] FIG. 12 TNF receptor assay of TAR1-5 19 homodimers in
different formats: dAb-linker-dAb format with 3U, 5U or 7U linker,
Fab format and cysteine hinge linker format.
[0207] FIG. 13 shows Dummy VH sequence for library 1. (amino acid
sequence ((SEQ ID NO:5; nucleotide sequences: coding strand (SEQ ID
NO:19), noncoding strand (SEQ ID NO:20). The sequence of the VH
framework based on germline sequence DP47-JH4b. Positions where NNK
randomisation (N=A or T or C or G nucleotides; K=G or T
nucleotides) has been incorporated into library 1 are indicated in
bold underlined text.
[0208] FIG. 14 shows Dummy VH sequence for library 2. (amino acid
sequence ((SEQ ID NO:21; nucleotide sequences: coding strand (SEQ
ID NO:22), noncoding strand (SEQ ID NO:23) The sequence of the VH
framework based on germline sequence DP47-JH4b. Positions where NNK
randomisation (N=A or T or C or G nucleotides; K=G or T
nucleotides) has been incorporated into library 2 are indicated in
bold underlined text.
[0209] FIG. 15 shows Dummy V.kappa. sequence for library 3. (amino
acid sequence ((SEQ ID NO:15; nucleotide sequences: coding strand
(SEQ ID NO:24), noncoding strand (SEQ ID NO:25) The sequence of the
V.kappa. framework based on germline sequence DP.sub.K9-J.sub.K1.
Positions where NNK randomisation (N=A or T or C or G nucleotides;
K=G or T nucleotides) has been incorporated into library 3 are
indicated in bold underlined text.
[0210] FIG. 16 shows nucleotide and amino acid sequence of anti MSA
dAbs MSA 16 (nucleotide sequence (SEQ ID NO:26), amino acid
sequence (SEQ ID NO:27)) and MSA 26 (nucleotide sequence (SEQ ID
NO:28), amino acid sequence (SEQ ID NO:29)).
[0211] FIG. 17 Inhibition biacore of MSA 16 and 26. Purified dAbs
MSA16 and MSA26 were analysed by inhibition biacore to determine
K.sub.d. Briefly, the dAbs were tested to determine the
concentration of dAb required to achieve 200RUs of response on a
biacore CM5 chip coated with a high density of MSA. Once the
required concentrations of dAb had been determined, MSA antigen at
a range of concentrations around the expected K.sub.d was premixed
with the dAb and incubated overnight. Binding to the MSA coated
biacore chip of dAb in each of the premixes was then measured at a
high flow-rate of 30 .mu.l/minute.
[0212] FIG. 18 Serum levels of MSA16 following injection. Serum
half life of the dAb MSA16 was determined in mouse. MSA16 was dosed
as single i.v. injections at approx 1.5 mg/kg into CD1 mice.
Modelling with a 2 compartment model showed MSA16 had a t1/2.alpha.
of 0.98 hr, a t1/2.beta. of 36.5 hr and an AUC of 913 hr.mg/ml.
MSA16 had a considerably lengthened half life compared with HEL4
(an anti-hen egg white lysozyme dAb) which had a t1/2.alpha. of
0.06 hr and a t1/2.beta. of 0.34 hr.
[0213] FIG. 19 ELISA (a) and TNF receptor assay (c) showing
inhibition of TNF binding with a Fab-like fragment comprising
MSA26Ck and TAR1-5-19CH. Addition of MSA with the Fab-like fragment
reduces the level of inhibition. An ELISA plate coated with 1
.mu.g/ml TNF.alpha. was probed with dual specific V.kappa. C.sub.H
and V.kappa. C.kappa. Fab like fragment and also with a control
TNF.alpha. binding dAb at a concentration calculated to give a
similar signal on the ELISA. Both the dual specific and control dAb
were used to probe the ELISA plate in the presence and in the
absence of 2 mg/ml MSA. The signal in the dual specific well was
reduced by more than 50% but the signal in the dAb well was not
reduced at all (see FIG. 19a). The same dual specific protein was
also put into the receptor assay with and without MSA and
competition by MSA was also shown (see FIG. 19c). This demonstrates
that binding of MSA to the dual specific is competitive with
binding to TNF.alpha.
[0214] FIG. 20 TNF receptor assay showing inhibiton of TNF binding
with a disulphide bonded heterodimer of TAR1-5-19 dAb and MSA16
dAb. Addition of MSA with the dimer reduces the level of inhibiton
in a dose dependant manner. The TNF receptor assay (FIG. 19(b)) was
conducted in the presence of a constant concentration of
heterodimer (18 nM) and a dilution series of MSA and HSA. The
presence of HSA at a range of concentrations (up to 2 mg/ml) did
not cause a reduction in the ability of the dimer to inhibit
TNF.alpha.. However, the addition of MSA caused a dose dependant
reduction in the ability of the dimer to inhibit TNF.alpha. (FIG.
19a). This demonstrates that MSA and TNF.alpha. compete for binding
to the cys bonded TAR1-5-19, MSA16 dimer. MSA and HSA alone did not
have an effect on the TNF binding level in the assay.
DETAILED DESCRIPTION OF THE INVENTION
[0215] Definitions
[0216] Complementary Two immunoglobulin domains are "complementary"
where they belong to families of structures which form cognate
pairs or groups or are derived from such families and retain this
feature. For example, a V.sub.H domain and a V.sub.L domain of an
antibody are complementary; two V.sub.H domains are not
complementary, and two V.sub.L domains are not complementary.
Complementary domains may be found in other members of the
immunoglobulin superfamily, such as the V.sub..alpha. and
V.sub..beta. (or .gamma. and .delta.) domains of the T-cell
receptor. In the context of the second configuration of the present
invention, non-complementary domains do not bind a target molecule
cooperatively, but act independently on different target epitopes
which may be on the same or different molecules. Domains which are
artificial, such as domains based on protein scaffolds which do not
bind epitopes unless engineered to do so, are non-complementary.
Likewise, two domains based on (for example) an immunoglobulin
domain and a fibronectin domain are not complementary.
[0217] Immunoglobulin This refers to a family of polypeptides which
retain the immunoglobulin fold characteristic of antibody
molecules, which contains two .beta. sheets and, usually, a
conserved disulphide bond. Members of the immunoglobulin
superfamily are involved in many aspects of cellular and
non-cellular interactions in vivo, including widespread roles in
the immune system (for example, antibodies, T-cell receptor
molecules and the like), involvement in cell adhesion (for example
the ICAM molecules) and intracellular signalling (for example,
receptor molecules, such as the PDGF receptor). The present
invention is applicable to all immunoglobulin superfamily molecules
which possess binding domains. Preferably, the present invention
relates to antibodies.
[0218] Combining Variable domains according to the invention are
combined to form a group of domains; for example, complementary
domains may be combined, such as V.sub.L domains being combined
with V.sub.H domains. Non-complementary domains may also be
combined. Domains may be combined in a number of ways, involving
linkage of the domains by covalent or non-covalent means.
[0219] Domain A domain is a folded protein structure which retains
its tertiary structure independently of the rest of the protein.
Generally, domains are responsible for discrete functional
properties of proteins, and in many cases may be added, removed or
transferred to other proteins without loss of function of the
remainder of the protein and/or of the domain. By single antibody
variable domain is meant a folded polypeptide domain comprising
sequences characteristic of antibody variable domains. It therefore
includes complete antibody variable domains and modified variable
domains, for example in which one or more loops have been replaced
by sequences which are not characteristic of antibody variable
domains, or antibody variable domains which have been truncated or
comprise N-- or C-terminal extensions, as well as folded fragments
of variable domains which retain at least in part the binding
activity and specificity of the full-length domain.
[0220] Repertoire A collection of diverse variants, for example
polypeptide variants which differ in their primary sequence. A
library used in the present invention will encompass a repertoire
of polypeptides comprising at least 1000 members.
[0221] Library The term library refers to a mixture of
heterogeneous polypeptides or nucleic acids. The library is
composed of members, each of which have a single polypeptide or
nucleic acid sequence. To this extent, library is synonymous with
repertoire. Sequence differences between library members are
responsible for the diversity present in the library. The library
may take the form of a simple mixture of polypeptides or nucleic
acids, or may be in the form of organisms or cells, for example
bacteria, viruses, animal or plant cells and the like, transformed
with a library of nucleic acids. Preferably, each individual
organism or cell contains only one or a limited number of library
members. Advantageously, the nucleic acids are incorporated into
expression vectors, in order to allow expression of the
polypeptides encoded by the nucleic acids. In a preferred aspect,
therefore, a library may take the form of a population of host
organisms, each organism containing one or more copies of an
expression vector containing a single member of the library in
nucleic acid form which can be expressed to produce its
corresponding polypeptide member. Thus, the population of host
organisms has the potential to encode a large repertoire of
genetically diverse polypeptide variants.
[0222] A `closed conformation multi-specific ligand` describes a
multi-specific ligand as herein defined comprising at least two
epitope binding domains as herein defined. The term `closed
conformation` (multi-specific ligand) means that the epitope
binding domains of the ligand are arranged such that epitope
binding by one epitope binding domain competes with epitope binding
by another epitope binding domain. That is, cognate epitopes may be
bound by each epitope binding domain individually but not
simultaneosuly. The closed conformation of the ligand can be
achieved using methods herein described.
[0223] Antibody An antibody (for example IgG, IgM, IgA, IgD or IgE)
or fragment (such as a Fab, F(ab').sub.2, Fv, disulphide linked Fv,
scFv, closed conformation multispecific antibody, disulphide-linked
scFv, diabody) whether derived from any species naturally producing
an antibody, or created by recombinant DNA technology; whether
isolated from serum, B-cells, hybridomas, transfectomas, yeast or
bacteria).
[0224] Dual-specific ligand A ligand comprising a first
immunoglobulin single variable domain and a second immunoglobulin
single variable domain as herein defined, wherein the variable
regions are capable of binding to two different antigens or two
epitopes on the same antigen which are not normally bound by a
monospecific immunoglobulin. For example, the two epitopes may be
on the same hapten, but are not the same epitope or sufficiently
adjacent to be bound by a monospecific ligand. The dual specific
ligands according to the invention are composed of variable domains
which have different specificities, and do not contain mutually
complementary variable domain pairs which have the same
specificity.
[0225] Antigen A molecule that is bound by a ligand according to
the present invention. Typically, antigens are bound by antibody
ligands and are capable of raising an antibody response in vivo. It
may be a polypeptide, protein, nucleic acid or other molecule.
Generally, the dual specific ligands according to the invention are
selected for target specificity against a particular antigen. In
the case of conventional antibodies and fragments thereof, the
antibody binding site defined by the variable loops (L1, L2, L3 and
H1, H2, H3) is capable of binding to the antigen.
[0226] Epitope A unit of structure conventionally bound by an
immunoglobulin V.sub.H/V.sub.L pair. Epitopes define the minimum
binding site for an antibody, and thus represent the target of
specificity of an antibody. In the case of a single domain
antibody, an epitope represents the unit of structure bound by a
variable domain in isolation.
[0227] Generic ligand A ligand that binds to all members of a
repertoire. Generally, not bound through the antigen binding site
as defined above. Non-limiting examples include protein A, protein
L and protein G.
[0228] Selecting Derived by screening, or derived by a Darwinian
selection process, in which binding interactions are made between a
domain and the antigen or epitope or between an antibody and an
antigen or epitope. Thus a first variable domain may be selected
for binding to an antigen or epitope in the presence or in the
absence of a complementary variable domain.
[0229] Universal framework A single antibody framework sequence
corresponding to the regions of an antibody conserved in sequence
as defined by Kabat ("Sequences of Proteins of Immunological
Interest", US Department of Health and Human Services) or
corresponding to the human germline immunoglobulin repertoire or
structure as defined by Chothia and Lesk, (1987) J. Mol. Biol.
196:910-917. The invention provides for the use of a single
framework, or a set of such frameworks, which has been found to
permit the derivation of virtually any binding specificity though
variation in the hypervariable regions alone.
[0230] Half-life The time taken for the serum concentration of the
ligand to reduce by 50%, in vivo, for example due to degradation of
the ligand and/or clearance or sequestration of the ligand by
natural mechanisms. The ligands of the invention are stabilised in
vivo and their half-life increased by binding to molecules which
resist degradation and/or clearance or sequestration. Typically,
such molecules are naturally occurring proteins which themselves
have a long half-life in vivo. The half-life of a ligand is
increased if its functional activity persists, in vivo, for a
longer period than a similar ligand which is not specific for the
half-life increasing molecule. Thus, a ligand specific for HSA and
a target molecule is compared with the same ligand wherein the
specificity for HSA is not present, that it does not bind HSA but
binds another molecule. For example, it may bind a second epitope
on the target molecule. Typically, the half life is increased by
10%, 20%, 30%, 40%, 50% or more. Increases in the range of
2.times., 3.times., 4.times., 5.times., 10.times., 20.times.,
30.times., 40.times., 50.times. or more of the half life are
possible. Alternatively, or in addition, increases in the range of
up to 30.times., 40.times., 50.times., 60.times., 70.times.,
80.times., 90.times., 100.times., 150.times. of the half life are
possible.
[0231] Homogeneous immunoassay An immunoassay in which analyte is
detected without need for a step of separating bound and un-bound
reagents.
[0232] Substantially identical (or "substantially homologous") A
first amino acid or nucleotide sequence that contains a sufficient
number of identical or equivalent (e.g., with a similar side chain,
e.g., conserved amino acid substitutions) amino acid residues or
nucleotides to a second amino acid or nucleotide sequence such that
the first and second amino acid or nucleotide sequences have
similar activities. In the case of antibodies, the second antibody
has the same binding specificity and has at least 50% of the
affinity of the same.
[0233] As used herein, the terms "low stringency," "medium
stringency," "high stringency," or "very high stringency
conditions" describe conditions for nucleic acid hybridization and
washing. Guidance for performing hybridization reactions can be
found in Current Protocols in Molecular Biology, John Wiley &
Sons, N.Y. (1989), 6.3.1-6.3.6, which is incorporated herein by
reference in its entirety. Aqueous and nonaqueous methods are
described in that reference and either can be used. Specific
hybridization conditions referred to herein are as follows: (1) low
stringency hybridization conditions in 6.times. sodium
chloride/sodium citrate (SSC) at about 45.degree. C., followed by
two washes in 0.2.times.SSC, 0.1% SDS at least at 50.degree. C.
(the temperature of the washes can be increased to 55.degree. C.
for low stringency conditions); (2) medium stringency hybridization
conditions in 6.times.SSC at about 45.degree. C., followed by one
or more washes in 0.2.times.SSC, 0.1% SDS at 60.degree. C.; (3)
high stringency hybridization conditions in 6.times.SSC at about
45.degree. C., followed by one or more washes in 0.2.times.SSC,
0.1% SDS at 65.degree. C.; and preferably (4) very high stringency
hybridization conditions are 0.5M sodium phosphate, 7% SDS at
65.degree. C., followed by one or more washes at 0.2.times.SSC, 1%
SDS at 65.degree. C. Very high stringency conditions (4) are the
preferred conditions and the ones that should be used unless
otherwise specified.
DETAILED DESCRIPTION OF THE INVENTION
[0234] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art (e.g., in cell culture, molecular
genetics, nucleic acid chemistry, hybridisation techniques and
biochemistry). Standard techniques are used for molecular, genetic
and biochemical methods (see generally, Sambrook et al., Molecular
Cloning: A Laboratory Manual, 2d ed. (1989) Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y. and Ausubel et al.,
Short Protocols in Molecular Biology (1999) 4.sup.th Ed, John Wiley
& Sons, Inc. which are incorporated herein by reference) and
chemical methods.
[0235] Preparation of Immunoglobulin Based Multi-Specific
Ligands
[0236] Dual specific ligands according to the invention, whether
open or closed in conformation according to the desired
configuration of the invention, may be prepared according to
previously established techniques, used in the field of antibody
engineering, for the preparation of scFv, "phage" antibodies and
other engineered antibody molecules. Techniques for the preparation
of antibodies, and in particular bispecific antibodies, are for
example described in the following reviews and the references cited
therein: Winter & Milstein, (1991) Nature 349:293-299;
Plueckthun (1992) Immunological Reviews 130:151-188; Wright et al.,
(1992) Crti. Rev. Immunol. 12:125-168; Holliger, P. & Winter,
G. (1993) Curr. Op. Biotechn. 4, 446-449; Carter, et al. (1995) J.
Hematother. 4, 463-470; Chester, K. A. & Hawkins, R. E. (1995)
Trends Biotechn. 13, 294-300; Hoogenboom, H. R. (1997) Nature
Biotechnol. 15, 125-126; Fearon, D. (1997) Nature Biotechnol. 15,
618-619; Pluckthun, A. & Pack, P. (1997) Immunotechnology 3,
83-105; Carter, P. & Merchant, A. M. (1997) Curr. Opin.
Biotechnol. 8, 449-454; Holliger, P. & Winter, G. (1997) Cancer
Immunol. Immunother. 45,128-130.
[0237] The invention provides for the selection of variable domains
against two different antigens or epitopes, and subsequent
combination of the variable domains.
[0238] The techniques employed for selection of the variable
domains employ libraries and selection procedures which are known
in the art. Natural libraries (Marks et al. (1991) J. Mol. Biol.,
222: 581; Vaughan et al. (1996) Nature Biotech., 14: 309) which use
rearranged V genes harvested from human B cells are well known to
those skilled in the art. Synthetic libraries (Hoogenboom &
Winter (1992) J. Mol. Biol., 227: 381; Barbas et al. (1992) Proc.
Natl. Acad. Sci. USA, 89: 4457; Nissim et al. (1994) EMBO J., 13:
692; Griffiths et al. (1994) EMBO J., 13: 3245; De Kruif et al.
(1995) J. Mol. Biol., 248: 97) are prepared by cloning
immunoglobulin V genes, usually using PCR. Errors in the PCR
process can lead to a high degree of randomisation. V.sub.H and/or
V.sub.L libraries may be selected against target antigens or
epitopes separately, in which case single domain binding is
directly selected for, or together.
[0239] A preferred method for making a dual specific ligand
according to the present invention comprises using a selection
system in which a repertoire of variable domains is selected for
binding to a first antigen or epitope and a repertoire of variable
domains is selected for binding to a second antigen or epitope. The
selected variable first and second variable domains are then
combined and the dual-specific ligand selected for binding to both
first and second antigen or epitope. Closed conformation ligands
are selected for binding both first and second antigen or epitope
in isolation but not simultaneously.
[0240] A. Library Vector Systems
[0241] A variety of selection systems are known in the art which
are suitable for use in the present invention. Examples of such
systems are described below.
[0242] Bacteriophage lambda expression systems may be screened
directly as bacteriophage plaques or as colonies of lysogens, both
as previously described (Huse et al. (1989) Science, 246: 1275;
Caton and Koprowski (1990) Proc. Natl. Acad. Sci. U.S.A., 87;
Mullinax et al. (1990) Proc. Natl. Acad. Sci. U.S.A., 87: 8095;
Persson et al. (1991) Proc. Natl. Acad. Sci. U.S.A., 88: 2432) and
are of use in the invention. Whilst such expression systems can be
used to screen up to 10.sup.6 different members of a library, they
are not really suited to screening of larger numbers (greater than
10.sup.6 members).
[0243] Of particular use in the construction of libraries are
selection display systems, which enable a nucleic acid to be linked
to the polypeptide it expresses. As used herein, a selection
display system is a system that permits the selection, by suitable
display means, of the individual members of the library by binding
the generic and/or target ligands.
[0244] Selection protocols for isolating desired members of large
libraries are known in the art, as typified by phage display
techniques. Such systems, in which diverse peptide sequences are
displayed on the surface of filamentous bacteriophage (Scott and
Smith (1990) Science, 249: 386), have proven useful for creating
libraries of antibody fragments (and the nucleotide sequences that
encoding them) for the in vitro selection and amplification of
specific antibody fragments that bind a target antigen (McCafferty
et al., WO 92/01047). The nucleotide sequences encoding the V.sub.H
and V.sub.L regions are linked to gene fragments which encode
leader signals that direct them to the periplasmic space of E. coli
and as a result the resultant antibody fragments are displayed on
the surface of the bacteriophage, typically as fusions to
bacteriophage coat proteins (e.g., pIII or pVIII). Alternatively,
antibody fragments are displayed externally on lambda phage capsids
(phagebodies). An advantage of phage-based display systems is that,
because they are biological systems, selected library members can
be amplified simply by growing the phage containing the selected
library member in bacterial cells. Furthermore, since the
nucleotide sequence that encode the polypeptide library member is
contained on a phage or phagemid vector, sequencing, expression and
subsequent genetic manipulation is relatively straightforward.
[0245] Methods for the construction of bacteriophage antibody
display libraries and lambda phage expression libraries are well
known in the art (McCafferty et al. (1990) Nature, 348: 552; Kang
et al. (1991) Proc. Natl. Acad. Sci. U.S.A., 88: 4363; Clackson et
al. (1991) Nature, 352: 624; Lowman et al. (1991) Biochemistry, 30:
10832; Burton et al. (1991) Proc. Natl. Acad. Sci U.S.A., 88:
10134; Hoogenboom et al. (1991) Nucleic Acids Res., 19: 4133; Chang
et al. (1991) J. Immunol., 147: 3610; Breitling et al. (1991) Gene,
104: 147; Marks et al. (1991) supra; Barbas et al. (1992) supra;
Hawkins and Winter (1992) J. Immunol., 22: 867; Marks et al., 1992,
J. Biol. Chem., 267: 16007; Lerner et al. (1992) Science, 258:
1313, incorporated herein by reference).
[0246] One particularly advantageous approach has been the use of
scFv phage-libraries (Huston et al., 1988, Proc. Natl. Acad. Sci
U.S.A., 85: 5879-5883; Chaudhary et al. (1990) Proc. Natl. Acad.
Sci U.S.A., 87: 1066-1070; McCafferty et al. (1990) supra; Clackson
et al. (1991) Nature, 352: 624; Marks et al. (1991) J. Mol. Biol.,
222: 581; Chiswell et al. (1992) Trends Biotech., 10: 80; Marks et
al. (1992) J. Biol. Chem., 267). Various embodiments of scFv
libraries displayed on bacteriophage coat proteins have been
described. Refinements of phage display approaches are also known,
for example as described in WO96/06213 and WO92/01047 (Medical
Research Council et al.) and WO97/08320 (Morphosys), which are
incorporated herein by reference.
[0247] Other systems for generating libraries of polypeptides
involve the use of cell-free enzymatic machinery for the in vitro
synthesis of the library members. In one method, RNA molecules are
selected by alternate rounds of selection against a target ligand
and PCR amplification (Tuerk and Gold (1990) Science, 249: 505;
Ellington and Szostak (1990) Nature, 346: 818). A similar technique
may be used to identify DNA sequences which bind a predetermined
human transcription factor (Thiesen and Bach (1990) Nucleic Acids
Res., 18: 3203; Beaudry and Joyce (1992) Science, 257: 635;
WO92/05258 and WO92/14843). In a similar way, in vitro translation
can be used to synthesise polypeptides as a method for generating
large libraries. These methods which generally comprise stabilised
polysome complexes, are described further in WO88/08453,
WO90/05785, WO90/07003, WO91/02076, WO91/05058, and WO92/02536.
Alternative display systems which are not phage-based, such as
those disclosed in WO95/22625 and WO95/11922 (Affymax) use the
polysomes to display polypeptides for selection.
[0248] A still further category of techniques involves the
selection of repertoires in artificial compartments, which allow
the linkage of a gene with its gene product. For example, a
selection system in which nucleic acids encoding desirable gene
products may be selected in microcapsules formed by water-in-oil
emulsions is described in WO99/02671, WO00/40712 and Tawfik &
Griffiths (1998) Nature Biotechnol 16(7), 652-6. Genetic elements
encoding a gene product having a desired activity are
compartmentalised into microcapsules and then transcribed and/or
translated to produce their respective gene products (RNA or
protein) within the microcapsules. Genetic elements which produce
gene product having desired activity are subsequently sorted. This
approach selects gene products of interest by detecting the desired
activity by a variety of means.
[0249] B. Library Construction.
[0250] Libraries intended for selection, may be constructed using
techniques known in the art, for example as set forth above, or may
be purchased from commercial sources. Libraries which are useful in
the present invention are described, for example, in WO99/20749.
Once a vector system is chosen and one or more nucleic acid
sequences encoding polypeptides of interest are cloned into the
library vector, one may generate diversity within the cloned
molecules by undertaking mutagenesis prior to expression;
alternatively, the encoded proteins may be expressed and selected,
as described above, before mutagenesis and additional rounds of
selection are performed. Mutagenesis of nucleic acid sequences
encoding structurally optimised polypeptides is carried out by
standard molecular methods. Of particular use is the polymerase
chain reaction, or PCR, (Mullis and Faloona (1987) Methods
Enzymol., 155: 335, herein incorporated by reference). PCR, which
uses multiple cycles of DNA replication catalysed by a
thermostable, DNA-dependent DNA polymerase to amplify the target
sequence of interest, is well known in the art. The construction of
various antibody libraries has been discussed in Winter et al.
(1994) Ann. Rev. Immunology 12, 433-55, and references cited
therein.
[0251] PCR is performed using template DNA (at least 1 fg; more
usefully, 1-1000 ng) and at least 25 pmol of oligonucleotide
primers; it may be advantageous to use a larger amount of primer
when the primer pool is heavily heterogeneous, as each sequence is
represented by only a small fraction of the molecules of the pool,
and amounts become limiting in the later amplification cycles. A
typical reaction mixture includes: 2 .mu.l of DNA, 25 pmol of
oligonucleotide primer, 2.5 .mu.l of 10.times. PCR buffer 1
(Perkin-Elmer, Foster City, Calif.), 0.4 .mu.l of 1.25 .mu.M dNTP,
0.15 .mu.l (or 2.5 units) of Taq DNA polymerase (Perkin Elmer,
Foster City, Calif.) and deionized water to a total volume of 25
.mu.l. Mineral oil is overlaid and the PCR is performed using a
programmable thermal cycler. The length and temperature of each
step of a PCR cycle, as well as the number of cycles, is adjusted
in accordance to the stringency requirements in effect. Annealing
temperature and timing are determined both by the efficiency with
which a primer is expected to anneal to a template and the degree
of mismatch that is to be tolerated; obviously, when nucleic acid
molecules are simultaneously amplified and mutagenised, mismatch is
required, at least in the first round of synthesis. The ability to
optimise the stringency of primer annealing conditions is well
within the knowledge of one of moderate skill in the art. An
annealing temperature of between 30.degree. C. and 72.degree. C. is
used. Initial denaturation of the template molecules normally
occurs at between 92.degree. C. and 99.degree. C. for 4 minutes,
followed by 20-40 cycles consisting of denaturation (94-99.degree.
C. for 15 seconds to 1 minute), annealing (temperature determined
as discussed above; 1-2 minutes), and extension (72.degree. C. for
1-5 minutes, depending on the length of the amplified product).
Final extension is generally for 4 minutes at 72.degree. C., and
may be followed by an indefinite (0-24 hour) step at 4.degree.
C.
[0252] C. Combining Single Variable Domains
[0253] Domains useful in the invention, once selected, may be
combined by a variety of methods known in the art, including
covalent and non-covalent methods.
[0254] Preferred methods include the use of polypeptide linkers, as
described, for example, in connection with scFv molecules (Bird et
al., (1988) Science 242:423-426). Discussion of suitable linkers is
provided in Bird et al. Science 242, 423-426; Hudson et al, Journal
Immunol Methods 231 (1999) 177-189; Hudson et al, Proc Nat Acad Sci
USA 85, 5879-5883. Linkers are preferably flexible, allowing the
two single domains to interact. One linker example is a (Gly.sub.4
Ser).sub.n linker, where n=1 to 8, eg, 2, 3, 4, 5 or 7. The linkers
used in diabodies, which are less flexible, may also be employed
(Holliger et al., (1993) PNAS (USA) 90:6444-6448).
[0255] In one embodiment, the linker employed is not an
immunoglobulin hinge region.
[0256] Variable domains may be combined using methods other than
linkers. For example, the use of disulphide bridges, provided
through naturally-occurring or engineered cysteine residues, may be
exploited to stabilise V.sub.H-V.sub.H,V.sub.L-V.sub.L or
V.sub.H-V.sub.L dimers (Reiter et al., (1994) Protein Eng.
7:697-704) or by remodelling the interface between the variable
domains to improve the "fit" and thus the stability of interaction
(Ridgeway et al., (1996) Protein Eng. 7:617-621; Zhu et al., (1997)
Protein Science 6:781-788).
[0257] Other techniques for joining or stabilising variable domains
of immunoglobulins, and in particular antibody V.sub.H domains, may
be employed as appropriate.
[0258] In accordance with the present invention, dual specific
ligands can be in "closed" conformations in solution. A "closed"
configuration is that in which the two domains (for example V.sub.H
and V.sub.L) are present in associated form, such as that of an
associated V.sub.H-V.sub.L pair which forms an antibody binding
site. For example, scFv may be in a closed conformation, depending
on the arrangement of the linker used to link the V.sub.H and
V.sub.L domains. If this is sufficiently flexible to allow the
domains to associate, or rigidly holds them in the associated
position, it is likely that the domains will adopt a closed
conformation.
[0259] Similarly, V.sub.H domain pairs and V.sub.L domain pairs may
exist in a closed conformation. Generally, this will be a function
of close association of the domains, such as by a rigid linker, in
the ligand molecule. Ligands in a closed conformation will be
unable to bind both the molecule which increases the half-life of
the ligand and a second target molecule. Thus, the ligand will
typically only bind the second target molecule on dissociation from
the molecule which increases the half-life of the ligand.
[0260] Moreover, the construction of V.sub.H/V.sub.H,
V.sub.L/V.sub.L or V.sub.H/V.sub.L dimers without linkers provides
for competition between the domains.
[0261] Ligands according to the invention may moreover be in an
open conformation. In such a conformation, the ligands will be able
to simultaneously bind both the molecule which increases the
half-life of the ligand and the second target molecule. Typically,
variable domains in an open configuration are (in the case of
V.sub.H-V.sub.L pairs) held far enough apart for the domains not to
interact and form an antibody binding site and not to compete for
binding to their respective epitopes. In the case of
V.sub.H/V.sub.H or V.sub.L/V.sub.L dimers, the domains are not
forced together by rigid linkers. Naturally, such domain pairings
will not compete for antigen binding or form an antibody binding
site.
[0262] Fab fragments and whole antibodies will exist primarily in
the closed conformation, although it will be appreciated that open
and closed dual specific ligands are likely to exist in a variety
of equilibria under different circumstances. Binding of the ligand
to a target is likely to shift the balance of the equilibrium
towards the open configuration. Thus, certain ligands according to
the invention can exist in two conformations in solution, one of
which (the open form) can bind two antigens or epitopes
independently, whilst the alternative conformation (the closed
form) can only bind one antigen or epitope; antigens or epitopes
thus compete for binding to the ligand in this conformation.
[0263] Although the open form of the dual specific ligand may thus
exist in equilibrium with the closed form in solution, it is
envisaged that the equilibrium will favour the closed form;
moreover, the open form can be sequestered by target binding into a
closed conformation. Preferably, therefore, certain dual specific
ligands of the invention are present in an equilibrium between two
(open and closed) conformations.
[0264] Dual specific ligands according to the invention may be
modified in order to favour an open or closed conformation. For
example, stabilisation of V.sub.H-V.sub.L interactions with
disulphide bonds stabilises the closed conformation. Moreover,
linkers used to join the domains, including V.sub.H domain and
V.sub.L domain pairs, may be constructed such that the open from is
favoured; for example, the linkers may sterically hinder the
association of the domains, such as by incorporation of large amino
acid residues in opportune locations, or the designing of a
suitable rigid structure which will keep the domains physically
spaced apart.
[0265] D. Characterisation of the Dual-Specific Ligand.
[0266] The binding of the dual-specific ligand to its specific
antigens or epitopes can be tested by methods which will be
familiar to those skilled in the art and include ELISA. In a
preferred embodiment of the invention binding is tested using
monoclonal phage ELISA.
[0267] Phage ELISA may be performed according to any suitable
procedure: an exemplary protocol is set forth below.
[0268] Populations of phage produced at each round of selection can
be screened for binding by ELISA to the selected antigen or
epitope, to identify "polyclonal" phage antibodies. Phage from
single infected bacterial colonies from these populations can then
be screened by ELISA to identify "monoclonal" phage antibodies. It
is also desirable to screen soluble antibody fragments for binding
to antigen or epitope, and this can also be undertaken by ELISA
using reagents, for example, against a C-- or N-terminal tag (see
for example Winter et al. (1994) Ann. Rev. Immunology 12, 433-55
and references cited therein.
[0269] The diversity of the selected phage monoclonal antibodies
may also be assessed by gel electrophoresis of PCR products (Marks
et al. 1991, supra; Nissim et al. 1994 supra), probing (Tomlinson
et al., 1992) J. Mol. Biol. 227, 776) or by sequencing of the
vector DNA.
[0270] E. Structure of `Dual-Specific Ligands`.
[0271] As described above, an antibody is herein defined as an
antibody (for example IgG, IgM, IgA, IgA, IgE) or fragment (Fab,
Fv, disulphide linked Fv, scFv, diabody) which comprises at least
one heavy and a light chain variable domain, at least two heavy
chain variable domains or at least two light chain variable
domains. It may be at least partly derived from any species
naturally producing an antibody, or created by recombinant DNA
technology; whether isolated from serum, B-cells, hybridomas,
transfectomas, yeast or bacteria).
[0272] In a preferred embodiment of the invention the dual-specific
ligand comprises at least one single heavy chain variable domain of
an antibody and one single light chain variable domain of an
antibody, or two single heavy or light chain variable domains. For
example, the ligand may comprise a V.sub.H/V.sub.L pair, a pair of
V.sub.H domains or a pair of V.sub.L domains.
[0273] The first and the second variable domains of such a ligand
may be on the same polypeptide chain. Alternatively they may be on
separate polypeptide chains. In the case that they are on the same
polypeptide chain they may be linked by a linker, which is
preferentially a peptide sequence, as described above.
[0274] The first and second variable domains may be covalently or
non-covalently associated. In the case that they are covalently
associated, the covalent bonds may be disulphide bonds.
[0275] In the case that the variable domains are selected from
V-gene repertoires selected for instance using phage display
technology as herein described, then these variable domains
comprise a universal framework region, such that is they may be
recognised by a specific generic ligand as herein defined. The use
of universal frameworks, generic ligands and the like is described
in WO99/20749.
[0276] Where V-gene repertoires are used variation in polypeptide
sequence is preferably located within the structural loops of the
variable domains. The polypeptide sequences of either variable
domain may be altered by DNA shuffling or by mutation in order to
enhance the interaction of each variable domain with its
complementary pair. DNA shuffling is known in the art and taught,
for example, by Stemmer, 1994, Nature 370: 389-391 and U.S. Pat.
No. 6,297,053, both of which are incorporated herein by reference.
Other methods of mutagenesis are well known to those of skill in
the art.
[0277] In a preferred embodiment of the invention the
`dual-specific ligand` is a single chain Fv fragment. In an
alternative embodiment of the invention, the `dual-specific ligand`
consists of a Fab format.
[0278] In a further aspect, the present invention provides nucleic
acid encoding at least a `dual-specific ligand` as herein
defined.
[0279] One skilled in the art will appreciate that, depending on
the aspect of the invention, both antigens or epitopes may bind
simultaneously to the same antibody molecule. Alternatively, they
may compete for binding to the same antibody molecule. For example,
where both epitopes are bound simultaneously, both variable domains
of a dual specific ligand are able to independently bind their
target epitopes. Where the domains compete, the one variable domain
is capable of binding its target, but not at the same time as the
other variable domain binds its cognate target; or the first
variable domain is capable of binding its target, but not at the
same time as the second variable domain binds its cognate
target.
[0280] The variable regions may be derived from antibodies directed
against target antigens or epitopes. Alternatively they may be
derived from a repertoire of single antibody domains such as those
expressed on the surface of filamentous bacteriophage. Selection
may be performed as described below.
[0281] In general, the nucleic acid molecules and vector constructs
required for the performance of the present invention may be
constructed and manipulated as set forth in standard laboratory
manuals, such as Sambrook et al. (1989) Molecular Cloning. A
Laboratory Manual, Cold Spring Harbor, USA.
[0282] The manipulation of nucleic acids useful in the present
invention is typically carried out in recombinant vectors.
[0283] Thus in a further aspect, the present invention provides a
vector comprising nucleic acid encoding at least a `dual-specific
ligand` as herein defined.
[0284] As used herein, vector refers to a discrete element that is
used to introduce heterologous DNA into cells for the expression
and/or replication thereof. Methods by which to select or construct
and, subsequently, use such vectors are well known to one of
ordinary skill in the art. Numerous vectors are publicly available,
including bacterial plasmids, bacteriophage, artificial chromosomes
and episomal vectors. Such vectors may be used for simple cloning
and mutagenesis; alternatively gene expression vector is employed.
A vector of use according to the invention may be selected to
accommodate a polypeptide coding sequence of a desired size,
typically from 0.25 kilobase (kb) to 40 kb or more in length A
suitable host cell is transformed with the vector after in vitro
cloning manipulations. Each vector contains various functional
components, which generally include a cloning (or "polylinker")
site, an origin of replication and at least one selectable marker
gene. If given vector is an expression vector, it additionally
possesses one or more of the following: enhancer element, promoter,
transcription termination and signal sequences, each positioned in
the vicinity of the cloning site, such that they are operatively
linked to the gene encoding a ligand according to the
invention.
[0285] Both cloning and expression vectors generally contain
nucleic acid sequences that enable the vector to replicate in one
or more selected host cells. Typically in cloning vectors, this
sequence is one that enables the vector to replicate independently
of the host chromosomal DNA and includes origins of replication or
autonomously replicating sequences. Such sequences are well known
for a variety of bacteria, yeast and viruses. The origin of
replication from the plasmid pBR322 is suitable for most
Gram-negative bacteria, the 2 micron plasmid origin is suitable for
yeast, and various viral origins (e.g. SV 40, adenovirus) are
useful for cloning vectors in mammalian cells. Generally, the
origin of replication is not needed for mammalian expression
vectors unless these are used in mammalian cells able to replicate
high levels of DNA, such as COS cells.
[0286] Advantageously, a cloning or expression vector may contain a
selection gene also referred to as selectable marker. This gene
encodes a protein necessary for the survival or growth of
transformed host cells grown in a selective culture medium. Host
cells not transformed with the vector containing the selection gene
will therefore not survive in the culture medium. Typical selection
genes encode proteins that confer resistance to antibiotics and
other toxins, e.g. ampicillin, neomycin, methotrexate or
tetracycline, complement auxotrophic deficiencies, or supply
critical nutrients not available in the growth media.
[0287] Since the replication of vectors encoding a ligand according
to the present invention is most conveniently performed in E. coli,
an E. coli-selectable marker, for example, the .beta.-lactamase
gene that confers resistance to the antibiotic ampicillin, is of
use. These can be obtained from E. coli plasmids, such as pBR322 or
a pUC plasmid such as pUC18 or pUC19.
[0288] Expression vectors usually contain a promoter that is
recognised by the host organism and is operably linked to the
coding sequence of interest. Such a promoter may be inducible or
constitutive. The term "operably linked" refers to a juxtaposition
wherein the components described are in a relationship permitting
them to function in their intended manner. A control sequence
"operably linked" to a coding sequence is ligated in such a way
that expression of the coding sequence is achieved under conditions
compatible with the control sequences.
[0289] Promoters suitable for use with prokaryotic hosts include,
for example, the .beta.-lactamase and lactose promoter systems,
alkaline phosphatase, the tryptophan (trp) promoter system and
hybrid promoters such as the tac promoter. Promoters for use in
bacterial systems will also generally contain a Shine-Delgarno
sequence operably linked to the coding sequence.
[0290] The preferred vectors are expression vectors that enables
the expression of a nucleotide sequence corresponding to a
polypeptide library member. Thus, selection with the first and/or
second antigen or epitope can be performed by separate propagation
and expression of a single clone expressing the polypeptide library
member or by use of any selection display system. As described
above, the preferred selection display system is bacteriophage
display. Thus, phage or phagemid vectors may be used, eg pIT1 or
pIT2. Leader sequences useful in the invention include pelB, stII,
ompA, phoA, bla and pelA. One example are phagemid vectors which
have an E. coli. origin of replication (for double stranded
replication) and also a phage origin of replication (for production
of single-stranded DNA). The manipulation and expression of such
vectors is well known in the art (Hoogenboom and Winter (1992)
supra; Nissim et al. (1994) supra). Briefly, the vector contains a
.beta.-lactamase gene to confer selectivity on the phagemid and a
lac promoter upstream of a expression cassette that consists (N to
C terminal) of a pelB leader sequence (which directs the expressed
polypeptide to the periplasmic space), a multiple cloning site (for
cloning the nucleotide version of the library member), optionally,
one or more peptide tag (for detection), optionally, one or more
TAG stop codon and the phage protein pIII. Thus, using various
suppressor and non-suppressor strains of E. coli and with the
addition of glucose, iso-propyl thio-.beta.-D-galactoside (IPTG) or
a helper phage, such as VCS M13, the vector is able to replicate as
a plasmid with no expression, produce large quantities of the
polypeptide library member only or produce phage, some of which
contain at least one copy of the polypeptide-pIII fusion on their
surface.
[0291] Construction of vectors encoding ligands according to the
invention employs conventional ligation techniques. Isolated
vectors or DNA fragments are cleaved, tailored, and religated in
the form desired to generate the required vector. If desired,
analysis to confirm that the correct sequences are present in the
constructed vector can be performed in a known fashion. Suitable
methods for constructing expression vectors, preparing in vitro
transcripts, introducing DNA into host cells, and performing
analyses for assessing expression and function are known to those
skilled in the art. The presence of a gene sequence in a sample is
detected, or its amplification and/or expression quantified by
conventional methods, such as Southern or Northern analysis,
Western blotting, dot blotting of DNA, RNA or protein, in situ
hybridisation, immunocytochemistry or sequence analysis of nucleic
acid or protein molecules. Those skilled in the art will readily
envisage how these methods may be modified, if desired.
[0292] Structure of Closed Conformation Multispecific Ligands
[0293] According to one aspect of the second configuration of the
invention present invention, the two or more non-complementary
epitope binding domains are linked so that they are in a closed
conformation as herein defined. Advantageously, they may be further
attached to a skeleton which may, as a alternative, or on addition
to a linker described herein, facilitate the formation and/or
maintenance of the closed conformation of the epitope binding sites
with respect to one another.
[0294] (I) Skeletons
[0295] Skeletons may be based on immunoglobulin molecules or may be
non-immunoglobulin in origin as set forth above. Preferred
immunoglobulin skeletons as herein defined includes any one or more
of those selected from the following: an immunoglobulin molecule
comprising at least (i) the CL (kappa or lambda subclass) domain of
an antibody; or (ii) the CH1 domain of an antibody heavy chain; an
immunoglobulin molecule comprising the CH1 and CH2 domains of an
antibody heavy chain; an immunoglobulin molecule comprising the
CH1, CH2 and CH3 domains of an antibody heavy chain; or any of the
subset (ii) in conjunction with the CL (kappa or lambda subclass)
domain of an antibody. A hinge region domain may also be included.
Such combinations of domains may, for example, mimic natural
antibodies, such as IgG or IgM, or fragments thereof, such as Fv,
scFv, Fab or F(ab').sub.2 molecules. Those skilled in the art will
be aware that this list is not intended to be exhaustive.
[0296] (II) Protein Scaffolds
[0297] Each epitope binding domain comprises a protein scaffold and
one or more CDRs which are involved in the specific interaction of
the domain with one or more epitopes. Advantageously, an epitope
binding domain according to the present invention comprises three
CDRs. Suitable protein scaffolds include any of those selected from
the group consisting of the following: those based on
immunoglobulin domains, those based on fibronectin, those based on
affibodies, those based on CTLA4, those based on chaperones such as
GroEL, those based on lipocallin and those based on the bacterial
Fc receptors SpA and SpD. Those skilled in the art will appreciate
that this list is not intended to be exhaustive.
[0298] F: Scaffolds for use in Constructing Dual Specific
Ligands
[0299] i. Selection of the Main-Chain Conformation
[0300] The members of the immunoglobulin superfamily all share a
similar fold for their polypeptide chain. For example, although
antibodies are highly diverse in terms of their primary sequence,
comparison of sequences and crystallographic structures has
revealed that, contrary to expectation, five of the six antigen
binding loops of antibodies (H1, H2, L1, L2, L3) adopt a limited
number of main-chain conformations, or canonical structures
(Chothia and Lesk (1987) J. Mol. Biol., 196: 901; Chothia et al.
(1989) Nature, 342: 877).
[0301] Analysis of loop lengths and key residues has therefore
enabled prediction of the main-chain conformations of H1, H2, L1,
L2 and L3 found in the majority of human antibodies (Chothia et al.
(1992) J. Mol. Biol., 227: 799; Tomlinson et al. (1995) EMBO J.,
14: 4628; Williams et al. (1996) J. Mol. Biol., 264: 220). Although
the H3 region is much more diverse in terms of sequence, length and
structure (due to the use of D segments), it also forms a limited
number of main-chain conformations for short loop lengths which
depend on the length and the presence of particular residues, or
types of residue, at key positions in the loop and the antibody
framework (Martin et al. (1996) J. Mol. Biol., 263: 800; Shirai et
al. (1996) FEBS Letters, 399: 1).
[0302] The dual specific ligands of the present invention are
advantageously assembled from libraries of domains, such as
libraries of V.sub.H domains and/or libraries of V.sub.L domains.
Moreover, the dual specific ligands of the invention may themselves
be provided in the form of libraries. In one aspect of the present
invention, libraries of dual specific ligands and/or domains are
designed in which certain loop lengths and key residues have been
chosen to ensure that the main-chain conformation of the members is
known. Advantageously, these are real conformations of
immunoglobulin superfamily molecules found in nature, to minimise
the chances that they are non-functional, as discussed above.
Germline V gene segments serve as one suitable basic framework for
constructing antibody or T-cell receptor libraries; other sequences
are also of use. Variations may occur at a low frequency, such that
a small number of functional members may possess an altered
main-chain conformation, which does not affect its function.
[0303] Canonical structure theory is also of use to assess the
number of different main-chain conformations encoded by ligands, to
predict the main-chain conformation based on ligand sequences and
to chose residues for diversification which do not affect the
canonical structure. It is known that, in the human V.sub..kappa.
domain, the L1 loop can adopt one of four canonical structures, the
L2 loop has a single canonical structure and that 90% of human
V.sub..kappa. domains adopt one of four or five canonical
structures for the L3 loop (Tomlinson et al. (1995) supra); thus,
in the V.sub..kappa. domain alone, different canonical structures
can combine to create a range of different main-chain
conformations. Given that the V.sub..lamda. domain encodes a
different range of canonical structures for the L1, L2 and L3 loops
and that V.sub..kappa. and V.sub..lamda. domains can pair with any
V.sub.H domain which can encode several canonical structures for
the H1 and H2 loops, the number of canonical structure combinations
observed for these five loops is very large. This implies that the
generation of diversity in the main-chain conformation may be
essential for the production of a wide range of binding
specificities. However, by constructing an antibody library based
on a single known main-chain conformation it has been found,
contrary to expectation, that diversity in the main-chain
conformation is not required to generate sufficient diversity to
target substantially all antigens. Even more surprisingly, the
single main-chain conformation need not be a consensus structure--a
single naturally occurring conformation can be used as the basis
for an entire library. Thus, in a preferred aspect, the
dual-specific ligands of the invention possess a single known
main-chain conformation.
[0304] The single main-chain conformation that is chosen is
preferably commonplace among molecules of the immunoglobulin
superfamily type in question. A conformation is commonplace when a
significant number of naturally occurring molecules are observed to
adopt it. Accordingly, in a preferred aspect of the invention, the
natural occurrence of the different main-chain conformations for
each binding loop of an immunoglobulin domain are considered
separately and then a naturally occurring variable domain is chosen
which possesses the desired main-chain conformations for the
different loops. If none is available, the nearest equivalent may
be chosen. It is preferable that the desired combination of
main-chain conformations for the different loops is created by
selecting germline gene segments which encode the desired
main-chain conformations. It is more preferable, that the selected
germline gene segments are frequently expressed in nature, and most
preferable that they are the most frequently expressed of all
natural germline gene segments.
[0305] In designing dual specific ligands or libraries thereof the
incidence of the different main-chain conformations for each of the
six antigen binding loops may be considered separately. For H1, H2,
L1, L2 and L3, a given conformation that is adopted by between 20%
and 100% of the antigen binding loops of naturally occurring
molecules is chosen. Typically, its observed incidence is above 35%
(i.e. between 35% and 100%) and, ideally, above 50% or even above
65%. Since the vast majority of H3 loops do not have canonical
structures, it is preferable to select a main-chain conformation
which is commonplace among those loops which do display canonical
structures. For each of the loops, the conformation which is
observed most often in the natural repertoire is therefore
selected. In human antibodies, the most popular canonical
structures (CS) for each loop are as follows: H1-CS 1 (79% of the
expressed repertoire), H2-CS 3 (46%), L1-CS 2 of V.sub..kappa.
(39%), L2-CS 1 (100%), L3-CS 1 of V.sub..kappa. (36%) (calculation
assumes a .kappa.:.lamda. ratio of 70:30, Hood et al. (1967) Cold
Spring Harbor Symp. Quant. Biol., 48: 133). For H3 loops that have
canonical structures, a CDR3 length (Kabat et al. (1991) Sequences
of proteins of immunological interest, U.S. Department of Health
and Human Services) of seven residues with a salt-bridge from
residue 94 to residue 101 appears to be the most common. There are
at least 16 human antibody sequences in the EMBL data library with
the required H3 length and key residues to form this conformation
and at least two crystallographic structures in the protein data
bank which can be used as a basis for antibody modelling (2cgr and
1tet). The most frequently expressed germline gene segments that
this combination of canonical structures are the V.sub.H segment
3-23 (DP-47), the J.sub.H segment JH4b, the V.sub..kappa. segment
O2/O12 (DPK9) and the J.sub..kappa. segment J.sub..kappa.1. V.sub.H
segments DP45 and DP38 are also suitable. These segments can
therefore be used in combination as a basis to construct a library
with the desired single main-chain conformation.
[0306] Alternatively, instead of choosing the single main-chain
conformation based on the natural occurrence of the different
main-chain conformations for each of the binding loops in
isolation, the natural occurrence of combinations of main-chain
conformations is used as the basis for choosing the single
main-chain conformation. In the case of antibodies, for example,
the natural occurrence of canonical structure combinations for any
two, three, four, five or for all six of the antigen binding loops
can be determined. Here, it is preferable that the chosen
conformation is commonplace in naturally occurring antibodies and
most preferable that it observed most frequently in the natural
repertoire. Thus, in human antibodies, for example, when natural
combinations of the five antigen binding loops, H1, H2, L1, L2 and
L3, are considered, the most frequent combination of canonical
structures is determined and then combined with the most popular
conformation for the H3 loop, as a basis for choosing the single
main-chain conformation.
[0307] ii. Diversification of the Canonical Sequence
[0308] Having selected several known main-chain conformations or,
preferably a single known main-chain conformation, dual specific
ligands according to the invention or libraries for use in the
invention can be constructed by varying the binding site of the
molecule in order to generate a repertoire with structural and/or
functional diversity. This means that variants are generated such
that they possess sufficient diversity in their structure and/or in
their function so that they are capable of providing a range of
activities.
[0309] The desired diversity is typically generated by varying the
selected molecule at one or more positions. The positions to be
changed can be chosen at random or are preferably selected. The
variation can then be achieved either by randomisation, during
which the resident amino acid is replaced by any amino acid or
analogue thereof, natural or synthetic, producing a very large
number of variants or by replacing the resident amino acid with one
or more of a defined subset of amino acids, producing a more
limited number of variants.
[0310] Various methods have been reported for introducing such
diversity. Error-prone PCR (Hawkins et al. (1992) J. Mol. Biol.,
226: 889), chemical mutagenesis (Deng et al. (1994) J. Biol. Chem.,
269: 9533) or bacterial mutator strains (Low et al. (1996) J. Mol.
Biol., 260: 359) can be used to introduce random mutations into the
genes that encode the molecule. Methods for mutating selected
positions are also well known in the art and include the use of
mismatched oligonucleotides or degenerate oligonucleotides, with or
without the use of PCR. For example, several synthetic antibody
libraries have been created by targeting mutations to the antigen
binding loops. The H3 region of a human tetanus toxoid-binding Fab
has been randomised to create a range of new binding specificities
(Barbas et al. (1992) Proc. Natl. Acad. Sci. USA, 89: 4457). Random
or semi-random H3 and L3 regions have been appended to germline V
gene segments to produce large libraries with unmutated framework
regions (Hoogenboom & Winter (1992) J. Mol. Biol., 227: 381;
Barbas et al. (1992) Proc. Natl. Acad. Sci. USA, 89: 4457; Nissim
et al. (1994) EMBO J., 13: 692; Griffiths et al. (1994) EMBO J.,
13: 3245; De Kruif et al. (1995) J. Mol. Biol., 248: 97). Such
diversification has been extended to include some or all of the
other antigen binding loops (Crameri et al. (1996) Nature Med., 2:
100; Riechmann et al. (1995) Bio/Technology, 13: 475; Morphosys,
WO97/08320, supra).
[0311] Since loop randomisation has the potential to create
approximately more than 10.sup.15 structures for H3 alone and a
similarly large number of variants for the other five loops, it is
not feasible using current transformation technology or even by
using cell free systems to produce a library representing all
possible combinations. For example, in one of the largest libraries
constructed to date, 6.times.10.sup.10 different antibodies, which
is only a fraction of the potential diversity for a library of this
design, were generated (Griffiths et al. (1994) supra).
[0312] In a preferred embodiment, only those residues which are
directly involved in creating or modifying the desired function of
the molecule are diversified. For many molecules, the function will
be to bind a target and therefore diversity should be concentrated
in the target binding site, while avoiding changing residues which
are crucial to the overall packing of the molecule or to
maintaining the chosen main-chain conformation.
[0313] Diversification of the Canonical Sequence as it Applies to
Antibody Domains
[0314] In the case of antibody dual-specific ligands, the binding
site for the target is most often the antigen binding site. Thus,
in a highly preferred aspect, the invention provides libraries of
or for the assembly of antibody dual-specific ligands in which only
those residues in the antigen binding site are varied. These
residues are extremely diverse in the human antibody repertoire and
are known to make contacts in high-resolution antibody/antigen
complexes. For example, in L2 it is known that positions 50 and 53
are diverse in naturally occurring antibodies and are observed to
make contact with the antigen. In contrast, the conventional
approach would have been to diversify all the residues in the
corresponding Complementarity Determining Region (CDR1) as defined
by Kabat et al. (1991, supra), some seven residues compared to the
two diversified in the library for use according to the invention.
This represents a significant improvement in terms of the
functional diversity required to create a range of antigen binding
specificities.
[0315] In nature, antibody diversity is the result of two
processes: somatic recombination of germline V, D and J gene
segments to create a naive primary repertoire (so called germline
and junctional diversity) and somatic hypermutation of the
resulting rearranged V genes. Analysis of human antibody sequences
has shown that diversity in the primary repertoire is focused at
the centre of the antigen binding site whereas somatic
hypermutation spreads diversity to regions at the periphery of the
antigen binding site that are highly conserved in the primary
repertoire (see Tomlinson et al. (1996) J. Mol. Biol., 256: 813).
This complementarity has probably evolved as an efficient strategy
for searching sequence space and, although apparently unique to
antibodies, it can easily be applied to other polypeptide
repertoires. The residues which are varied are a subset of those
that form the binding site for the target. Different (including
overlapping) subsets of residues in the target binding site are
diversified at different stages during selection, if desired.
[0316] In the case of an antibody repertoire, an initial `naive`
repertoire is created where some, but not all, of the residues in
the antigen binding site are diversified. As used herein in this
context, the term "naive" refers to antibody molecules that have no
pre-determined target. These molecules resemble those which are
encoded by the immunoglobulin genes of an individual who has not
undergone immune diversification, as is the case with fetal and
newborn individuals, whose immune systems have not yet been
challenged by a wide variety of antigenic stimuli. This repertoire
is then selected against a range of antigens or epitopes. If
required, further diversity can then be introduced outside the
region diversified in the initial repertoire. This matured
repertoire can be selected for modified function, specificity or
affinity.
[0317] The invention provides two different naive repertoires of
binding domains for the construction of dual specific ligands, or a
naive library of dual specific ligands, in which some or all of the
residues in the antigen binding site are varied. The "primary"
library mimics the natural primary repertoire, with diversity
restricted to residues at the centre of the antigen binding site
that are diverse in the germline V gene segments (germline
diversity) or diversified during the recombination process
(junctional diversity). Those residues which are diversified
include, but are not limited to, H50, H52, H52a, H53, H55, H56,
H58, H95, H96, H97, H98, L50, L53, L91, L92, L93, L94 and L96. In
the "somatic" library, diversity is restricted to residues that are
diversified during the recombination process (junctional diversity)
or are highly somatically mutated). Those residues which are
diversified include, but are not limited to: H31, H33, H35, H95,
H96, H97, H98, L30, L31, L32, L34 and L96. All the residues listed
above as suitable for diversification in these libraries are known
to make contacts in one or more antibody-antigen complexes. Since
in both libraries, not all of the residues in the antigen binding
site are varied, additional diversity is incorporated during
selection by varying the remaining residues, if it is desired to do
so. It shall be apparent to one skilled in the art that any subset
of any of these residues (or additional residues which comprise the
antigen binding site) can be used for the initial and/or subsequent
diversification of the antigen binding site.
[0318] In the construction of libraries for use in the invention,
diversification of chosen positions is typically achieved at the
nucleic acid level, by altering the coding sequence which specifies
the sequence of the polypeptide such that a number of possible
amino acids (all 20 or a subset thereof) can be incorporated at
that position. Using the IUPAC nomenclature, the most versatile
codon is NNK, which encodes all amino acids as well as the TAG stop
codon. The NNK codon is preferably used in order to introduce the
required diversity. Other codons which achieve the same ends are
also of use, including the NNN codon, which leads to the production
of the additional stop codons TGA and TAA.
[0319] A feature of side-chain diversity in the antigen binding
site of human antibodies is a pronounced bias which favours certain
amino acid residues. If the amino acid composition of the ten most
diverse positions in each of the V.sub.H, V.sub..kappa. and
V.sub..lamda. regions are summed, more than 76% of the side-chain
diversity comes from only seven different residues, these being,
serine (24%), tyrosine (14%), asparagine (11%), glycine (9%),
alanine (7%), aspartate (6%) and threonine (6%). This bias towards
hydrophilic residues and small residues which can provide
main-chain flexibility probably reflects the evolution of surfaces
which are predisposed to binding a wide range of antigens or
epitopes and may help to explain the required promiscuity of
antibodies in the primary repertoire.
[0320] Since it is preferable to mimic this distribution of amino
acids, the distribution of amino acids at the positions to be
varied preferably mimics that seen in the antigen binding site of
antibodies. Such bias in the substitution of amino acids that
permits selection of certain polypeptides (not just antibody
polypeptides) against a range of target antigens is easily applied
to any polypeptide repertoire. There are various methods for
biasing the amino acid distribution at the position to be varied
(including the use of tri-nucleotide mutagenesis, see WO97/08320),
of which the preferred method, due to ease of synthesis, is the use
of conventional degenerate codons. By comparing the amino acid
profile encoded by all combinations of degenerate codons (with
single, double, triple and quadruple degeneracy in equal ratios at
each position) with the natural amino acid use it is possible to
calculate the most representative codon. The codons (AGT)(AGC)T,
(AGT)(AGC)C and (AGT)(AGC)(CT)--that is, DVT, DVC and DVY,
respectively using IUPAC nomenclature--are those closest to the
desired amino acid profile: they encode 22% serine and 11%
tyrosine, asparagine, glycine, alanine, aspartate, threonine and
cysteine. Preferably, therefore, libraries are constructed using
either the DVT, DVC or DVY codon at each of the diversified
positions.
[0321] G: Antigens Capable of Increasing Ligand Half-Life
[0322] The dual specific ligands according to the invention, in one
configuration thereof, are capable of binding to one or more
molecules which can increase the half-life of the ligand in vivo.
Typically, such molecules are polypeptides which occur naturally in
vivo and which resist degradation or removal by endogenous
mechanisms which remove unwanted material from the organism. For
example, the molecule which increases the half-life of the organism
may be selected from the following:
[0323] Proteins from the extracellular matrix; for example
collagen, laminins, integrins and fibronectin. Collagens are the
major proteins of the extracellular matrix. About 15 types of
collagen molecules are currently known, found in different parts of
the body, eg type I collagen (accounting for 90% of body collagen)
found in bone, skin, tendon, ligaments, cornea, internal organs or
type II collagen found in cartilage, invertebral disc, notochord,
vitreous humour of the eye.
[0324] Proteins found in blood, including:
[0325] Plasma proteins such as fibrin, .alpha.-2 macroglobulin,
serum albumin, fibrinogen A, fibrinogen B, serum amyloid protein A,
heptaglobin, profilin, ubiquitin, uteroglobulin and
.beta.-2-microglobulin;
[0326] Enzymes and inhibitors such as plasminogen, lysozyme,
cystatin C, alpha-1-antitrypsin and pancreatic trypsin inhibitor.
Plasminogen is the inactive precursor of the trypsin-like serine
protease plasmin. It is normally found circulating through the
blood stream. When plasminogen becomes activated and is converted
to plasmin, it unfolds a potent enzymatic domain that dissolves the
fibrinogen fibers that entgangle the blood cells in a blood clot.
This is called fibrinolysis.
[0327] Immune system proteins, such as IgE, IgG, IgM.
[0328] Transport proteins such as retinol binding protein,
.alpha.-1 microglobulin.
[0329] Defensins such as beta-defensin 1, Neutrophil defensins 1,2
and 3.
[0330] Proteins found at the blood brain barrier or in neural
tissues, such as melanocortin receptor, myelin, ascorbate
transporter.
[0331] Transferrin receptor specific ligand-neuropharmaceutical
agent fusion proteins (see U.S. Pat. No. 5,977,307);
[0332] brain capillary endothelial cell receptor, transferrin,
transferrin receptor, insulin, insulin-like growth factor 1 (IGF 1)
receptor, insulin-like growth factor 2 (IGF 2) receptor, insulin
receptor.
[0333] Proteins localised to the kidney, such as polycystin, type
IV collagen, organic anion transporter K1, Heymann's antigen.
[0334] Proteins localised to the liver, for example alcohol
dehydrogenase, G250.
[0335] Blood coagulation factor X
[0336] .alpha.1 antitrypsin
[0337] HNF 1.alpha.
[0338] Proteins localised to the lung, such as secretory component
(binds IgA).
[0339] Proteins localised to the Heart, for example HSP 27. This is
associated with dilated cardiomyopathy.
[0340] Proteins localised to the skin, for example keratin.
[0341] Bone specific proteins, such as bone morphogenic proteins
(BMPs), which are a subset of the transforming growth factor .beta.
superfamily that demonstrate osteogenic activity. Examples include
BMP-2, -4, -5, -6, -7 (also referred to as osteogenic protein
(OP-1) and -8 (OP-2).
[0342] Tumour specific proteins, including human trophoblast
antigen, herceptin receptor, oestrogen receptor, cathepsins eg
cathepsin B (found in liver and spleen).
[0343] Disease-specific proteins, such as antigens expressed only
on activated T-cells: including LAG-3 (lymphocyte activation gene),
osteoprotegerin ligand (OPGL) see Nature 402, 304-309; 1999, OX40
(a member of the TNF receptor family, expressed on activated T
cells and the only costimulatory T cell molecule known to be
specifically up-regulated in human T cell leukaemia virus type-I
(HTLV-I)-producing cells.) See J Immunol. Jul. 1,
2000;165(1):263-70; Metalloproteases (associated with
arthritis/cancers), including CG6512 Drosophila, human paraplegin,
human FtsH, human AFG3L2, murine ftsH; angiogenic growth factors,
including acidic fibroblast growth factor (FGF-1), basic fibroblast
growth factor (FGF-2), Vascular endothelial growth factor/vascular
permeability factor (VEGF/VPF), transforming growth factor-a (TGF
a), tumor necrosis factor-alpha (TNF-.alpha.), angiogenin,
interleukin-3 (IL-3), interleukin-8 (IL-8), platelet-derived
endothelial growth factor (PD-ECGF), placental growth factor
(PIGF), midkine platelet-derived growth factor-BB (PDGF),
fractalkine.
[0344] Stress Proteins (Heat Shock Proteins)
[0345] HSPs are normally found intracellularly. When they are found
extracellularly, it is an indicator that a cell has died and
spilled out its contents. This unprogrammed cell death (necrosis)
only occurs when as a result of trauma, disease or injury and
therefore in vivo, extracellular HSPs trigger a response from the
immune system that will fight infection and disease. A dual
specific which binds to extracellular HSP can be localised to a
disease site.
[0346] Proteins involved in Fc transport
[0347] Brambell receptor (also known as FcRB)
[0348] This Fc receptor has two functions, both of which are
potentially useful for delivery
[0349] The functions are [0350] (1) The transport of IgG from
mother to child across the placenta [0351] (2) the protection of
IgG from degradation thereby prolonging its serum half life of IgG.
It is thought that the receptor recycles IgG from endosome.
[0352] See Holliger et al, Nat Biotechnol Jul. 15,
1997(7):632-6.
[0353] Ligands according to the invention may designed to be
specific for the above targets without requiring any increase in or
increasing half life in vivo. For example, ligands according to the
invention can be specific for targets selected from the foregoing
which are tissue-specific, thereby enabling tissue-specific
targeting of the dual specific ligand, or a dAb monomer that binds
a tissue-specific therapeutically relevant target, irrespective of
any increase in half-life, although this may result. Moreover,
where the ligand or dAb monomer targets kidney or liver, this may
redirect the ligand or dAb monomer to an alternative clearance
pathway in vivo (for example, the ligand may be directed away from
liver clearance to kidney clearance).
[0354] H: Use of Multispecific Ligands According to the Second
Configuration of the Invention
[0355] Multispecific ligands according to the method of the second
configuration of the present invention may be employed in in vivo
therapeutic and prophylactic applications, in vitro and in vivo
diagnostic applications, in vitro assay and reagent applications,
and the like. For example antibody molecules may be used in
antibody based assay techniques, such as ELISA techniques,
according to methods known to those skilled in the art.
[0356] As alluded to above, the multispecific ligands according to
the invention are of use in diagnostic, prophylactic and
therapeutic procedures. Multispecific antibodies according to the
invention are of use diagnostically in Western analysis and in situ
protein detection by standard immunohistochemical procedures; for
use in these applications, the ligands may be labelled in
accordance with techniques known to the art. In addition, such
antibody polypeptides may be used preparatively in affinity
chromatography procedures, when complexed to a chromatographic
support, such as a resin. All such techniques are well known to one
of skill in the art.
[0357] Diagnostic uses of the closed conformation multispecific
ligands according to the invention include homogenous assays for
analytes which exploit the ability of closed conformation
multispecific ligands to bind two targets in competition, such that
two targets cannot bind simultaneously (a closed conformation), or
alternatively their ability to bind two targets simultaneously (an
open conformation).
[0358] A true homogenous immunoassay format has been avidly sought
by manufacturers of diagnostics and research assay systems used in
drug discovery and development. The main diagnostics markets
include human testing in hospitals, doctor's offices and clinics,
commercial reference laboratories, blood banks, and the home,
non-human diagnostics (for example food testing, water testing,
environmental testing, bio-defence, and veterinary testing), and
finally research (including drug development; basic research and
academic research).
[0359] At present all these markets utilise immunoassay systems
that are built around chemiluminescent, ELISA, fluorescence or in
rare cases radio-immunoassay technologies. Each of these assay
formats requires a separation step (separating bound from un-bound
reagents). In some cases, several separation steps are required.
Adding these additional steps adds reagents and automation, takes
time, and affects the ultimate outcome of the assays. In human
diagnostics, the separation step may be automated, which masks the
problem, but does not remove it. The robotics, additional reagents,
additional incubation times, and the like add considerable cost and
complexity. In drug development, such as high throughput screening,
where literally millions of samples are tested at once, with very
low levels of test molecule, adding additional separation steps can
eliminate the ability to perform a screen. However, avoiding the
separation creates too much noise in the read out. Thus, there is a
need for a true homogenous format that provides sensitivities at
the range obtainable from present assay formats. Advantageously, an
assay possesses fully quantitative read-outs with high sensitivity
and a large dynamic range. Sensitivity is an important requirement,
as is reducing the amount of sample required. Both of these
features are features that a homogenous system offers. This is very
important in point of care testing, and in drug development where
samples are precious. Heterogenous systems, as currently available
in the art, require large quantities of sample and expensive
reagents
[0360] Applications for homogenous assays include cancer testing,
where the biggest assay is that for Prostate Specific Antigen, used
in screening men for prostate cancer. Other applications include
fertility testing, which provides a series of tests for women
attempting to conceive including beta-hcg for pregnancy. Tests for
infectious diseases, including hepatitis, HIV, rubella, and other
viruses and microorganisms and sexually transmitted diseases. Tests
are used by blood banks, especially tests for HIV, hepatitis A, B,
C, non A non B. Therapeutic drug monitoring tests include
monitoring levels of prescribed drugs in patients for efficacy and
to avoid toxicity, for example digoxin for arrhythmia, and
phenobarbital levels in psychotic cases; theophylline for asthma.
Diagnostic tests are moreover useful in abused drug testing, such
as testing for cocaine, marijuana and the like. Metabolic tests are
used for measuring thyroid function, anaemia and other
physiological disorders and functions.
[0361] The homogenous immunoassay format is moreover useful in the
manufacture of standard clinical chemistry assays. The inclusion of
immunoassays and chemistry assays on the same instrument is highly
advantageous in diagnostic testing. Suitable chemical assays
include tests for glucose, cholesterol, potassium, and the
like.
[0362] A further major application for homogenous immunoassays is
drug discovery and development: high throughput screening includes
testing combinatorial chemistry libraries versus targets in ultra
high volume. Signal is detected, and positive groups then split
into smaller groups, and eventually tested in cells and then
animals. Homogenous assays may be used in all these types of test.
In drug development, especially animal studies and clinical trials
heavy use of immunoassays is made. Homogenous assays greatly
accelerate and simplify these procedures. Other Applications
include food and beverage testing: testing meat and other foods for
E. coli, salmonella, etc; water testing, including testing at water
plants for all types of contaminants including E. coli; and
veterinary testing.
[0363] In a broad embodiment, the invention provides a binding
assay comprising a detectable agent which is bound to a closed
conformation multispecific ligand according to the invention, and
whose detectable properties are altered by the binding of an
analyte to said closed conformation multispecific ligand. Such an
assay may be configured in several different ways, each exploiting
the above properties of closed conformation multispecific
ligands.
[0364] The assay relies on the direct or indirect displacement of
an agent by the analyte, resulting in a change in the detectable
properties of the agent. For example, where the agent is an enzyme
which is capable of catalysing a reaction which has a detectable
end-point, said enzyme can be bound by the ligand such as to
obstruct its active site, thereby inactivating the enzyme. The
analyte, which is also bound by the closed conformation
multispecific ligand, displaces the enzyme, rendering it active
through freeing of the active site. The enzyme is then able to
react with a substrate, to give rise to a detectable event. In an
alternative embodiment, the ligand may bind the enzyme outside of
the active site, influencing the conformation of the enzyme and
thus altering its activity. For example, the structure of the
active site may be constrained by the binding of the ligand, or the
binding of cofactors necessary for activity may be prevented.
[0365] The physical implementation of the assay may take any form
known in the art. For example, the closed conformation
multispecific ligand/enzyme complex may be provided on a test
strip; the substrate may be provided in a different region of the
test strip, and a solvent containing the analyte allowed to migrate
through the ligand/enzyme complex, displacing the enzyme, and
carrying it to the substrate region to produce a signal.
Alternatively, the ligand/enzyme complex may be provided on a test
stick or other solid phase, and dipped into an analyte/substrate
solution, releasing enzyme into the solution in response to the
presence of analyte.
[0366] Since each molecule of analyte potentially releases one
enzyme molecule, the assay is quantitative, with the strength of
the signal generated in a given time being dependent on the
concentration of analyte in the solution.
[0367] Further configurations using the analyte in a closed
conformation are possible. For example, the closed conformation
multispecific ligand may be configured to bind an enzyme in an
allosteric site, thereby activating the enzyme. In such an
embodiment, the enzyme is active in the absence of analyte.
Addition of the analyte displaces the enzyme and removes allosteric
activation, thus inactivating the enzyme.
[0368] In the context of the above embodiments which employ enzyme
activity as a measure of the analyte concentration, activation or
inactivation of the enzyme refers to an increase or decrease in the
activity of the enzyme, measured as the ability of the enzyme to
catalyse a signal-generating reaction. For example, the enzyme may
catalyse the conversion of an undetectable substrate to a
detectable form thereof. For example, horseradish peroxidase is
widely used in the art together with chromogenic or
chemiluminescent substrates, which are available commercially. The
level of increase or decrease of the activity of the enzyme may
between 10% and 100%, such as 20%, 30%, 40%, 50%, 60%, 70%, 80% or
90%; in the case of an increase in activity, the increase may be
more than 100%, i.e. 200%, 300%, 500% or more, or may not be
measurable as a percentage if the baseline activity of the
inhibited enzyme is undetectable.
[0369] In a further configuration, the closed conformation
multispecific ligand may bind the substrate of an enzyme/substrate
pair, rather than the enzyme. The substrate is therefore
unavailable to the enzyme until released from the closed
conformation multispecific ligand through binding of the analyte.
The implementations for this configuration are as for the
configurations which bind enzyme.
[0370] Moreover, the assay may be configured to bind a fluorescent
molecule, such as a fluorescein or another fluorophore, in a
conformation such that the fluorescence is quenched on binding to
the ligand. In this case, binding of the analyte to the ligand will
displace the fluorescent molecule, thus producing a signal.
Alternatives to fluorescent molecules which are useful in the
present invention include luminescent agents, such as
luciferin/luciferase, and chromogenic agents, including agents
commonly used in immunoassays such as HRP.
[0371] Therapeutic and prophylactic uses of multispecific ligands
prepared according to the invention involve the administration of
ligands according to the invention to a recipient mammal, such as a
human. Multi-specificity can allow antibodies to bind to multimeric
antigen with great avidity. Multispecific ligands can allow the
cross-linking of two antigens, for example in recruiting cytotoxic
T-cells to mediate the killing of tumour cell lines.
[0372] Substantially pure ligands or binding proteins thereof, for
example dAb monomers, of at least 90 to 95% homogeneity are
preferred for administration to a mammal, and 98 to 99% or more
homogeneity is most preferred for pharmaceutical uses, especially
when the mammal is a human. Once purified, partially or to
homogeneity as desired, the ligands may be used diagnostically or
therapeutically (including extracorporeally) or in developing and
performing assay procedures, immunofluorescent stainings and the
like (Lefkovite and Pernis, (1979 and 1981) Immunological Methods,
Volumes I and II, Academic Press, NY).
[0373] The ligands or binding proteins thereof, for example dAb
monomers, of the present invention will typically find use in
preventing, suppressing or treating inflammatory states, allergic
hypersensitivity, cancer, bacterial or viral infection, and
autoimmune disorders (which include, but are not limited to, Type I
diabetes, asthma, multiple sclerosis, rheumatoid arthritis,
systemic lupus erythematosus, Crohn's disease and myasthenia
gravis).
[0374] In the instant application, the term "prevention" involves
administration of the protective composition prior to the induction
of the disease. "Suppression" refers to administration of the
composition after an inductive event, but prior to the clinical
appearance of the disease. "Treatment" involves administration of
the protective composition after disease symptoms become
manifest.
[0375] Animal model systems which can be used to screen the
effectiveness of the antibodies or binding proteins thereof in
protecting against or treating the disease are available. Methods
for the testing of systemic lupus erythematosus (SLE) in
susceptible mice are known in the art (Knight et al. (1978) J. Exp.
Med., 147: 1653; Reinersten et al. (1978) New Eng. J. Med., 299:
515). Myasthenia Gravis (MG) is tested in SJL/J female mice by
inducing the disease with soluble AchR protein from another species
(Lindstrom et al. (1988) Adv. Immunol., 42: 233). Arthritis is
induced in a susceptible strain of mice by injection of Type II
collagen (Stuart et al. (1984) Ann. Rev. Immunol., 42: 233). A
model by which adjuvant arthritis is induced in susceptible rats by
injection of mycobacterial heat shock protein has been described
(Van Eden et al. (1988) Nature, 331: 171). Thyroiditis is induced
in mice by administration of thyroglobulin as described (Maron et
al. (1980) J. Exp. Med., 152: 1115). Insulin dependent diabetes
mellitus (IDDM) occurs naturally or can be induced in certain
strains of mice such as those described by Kanasawa et al. (1984)
Diabetologia, 27: 113. EAE in mouse and rat serves as a model for
MS in human. In this model, the demyelinating disease is induced by
administration of myelin basic protein (see Paterson (1986)
Textbook of Immunopathology, Mischer et al., eds., Grune and
Stratton, New York, pp. 179-213; McFarlin et al. (1973) Science,
179:478: and Satoh et al. (1987) J. Immunol., 138: 179).
[0376] Generally, the present ligands will be utilised in purified
form together with pharmacologically appropriate carriers.
Typically, these carriers include aqueous or alcoholic/aqueous
solutions, emulsions or suspensions, any including saline and/or
buffered media. Parenteral vehicles include sodium chloride
solution, Ringer's dextrose, dextrose and sodium chloride and
lactated Ringer's. Suitable physiologically-acceptable adjuvants,
if necessary to keep a polypeptide complex in suspension, may be
chosen from thickeners such as carboxymethylcellulose,
polyvinylpyrrolidone, gelatin and alginates.
[0377] Intravenous vehicles include fluid and nutrient replenishers
and electrolyte replenishers, such as those based on Ringer's
dextrose. Preservatives and other additives, such as
antimicrobials, antioxidants, chelating agents and inert gases, may
also be present (Mack (1982) Remington's Pharmaceutical Sciences,
16th Edition).
[0378] The ligands of the present invention may be used as
separately administered compositions or in conjunction with other
agents. These can include various immunotherapeutic drugs, such as
cylcosporine, methotrexate, adriamycin or cisplatinum, and
immunotoxins. Pharmaceutical compositions can include "cocktails"
of various cytotoxic or other agents in conjunction with the
ligands of the present invention, or even combinations of lignds
according to the present invention having different specificities,
such as ligands selected using different target antigens or
epitopes, whether or not they are pooled prior to
administration.
[0379] The route of administration of pharmaceutical compositions
according to the invention may be any of those commonly known to
those of ordinary skill in the art. For therapy, including without
limitation immunotherapy, the selected ligands thereof of the
invention can be administered to any patient in accordance with
standard techniques. The administration can be by any appropriate
mode, including parenterally, intravenously, intramuscularly,
intraperitoneally, transdermally, via the pulmonary route, or also,
appropriately, by direct infusion with a catheter. The dosage and
frequency of administration will depend on the age, sex and
condition of the patient, concurrent administration of other drugs,
counterindications and other parameters to be taken into account by
the clinician.
[0380] The ligands of this invention can be lyophilised for storage
and reconstituted in a suitable carrier prior to use. This
technique has been shown to be effective with conventional
immunoglobulins and art-known lyophilisation and reconstitution
techniques can be employed. It will be appreciated by those skilled
in the art that lyophilisation and reconstitution can lead to
varying degrees of antibody activity loss (e.g. with conventional
immunoglobulins, IgM antibodies tend to have greater activity loss
than IgG antibodies) and that use levels may have to be adjusted
upward to compensate.
[0381] The compositions containing the present ligands or a
cocktail thereof can be administered for prophylactic and/or
therapeutic treatments. In certain therapeutic applications, an
adequate amount to accomplish at least partial inhibition,
suppression, modulation, killing, or some other measurable
parameter, of a population of selected cells is defined as a
"therapeutically-effective dose". Amounts needed to achieve this
dosage will depend upon the severity of the disease and the general
state of the patient's own immune system, but generally range from
0.005 to 5.0 mg of ligand, e.g. antibody, receptor (e.g. a T-cell
receptor) or binding protein thereof per kilogram of body weight,
with doses of 0.05 to 2.0 mg/kg/dose being more commonly used. For
prophylactic applications, compositions containing the present
ligands or cocktails thereof may also be administered in similar or
slightly lower dosages.
[0382] Treatment performed using the compositions described herein
is considered "effective" if one or more symptoms is reduced (e.g.,
by at least 10% or at least one point on a clinical assessment
scale), relative to such symptoms present before treatment, or
relative to such symptoms in an individual (human or model animal)
not treated with such composition. Symptoms will obviously vary
depending upon the disease or disorder targeted, but can be
measured by an ordinarily skilled clinician or technician. Such
symptoms can be measured, for example, by monitoring the level of
one or more biochemical indicators of the disease or disorder
(e.g., levels of an enzyme or metabolite correlated with the
disease, affected cell numbers, etc.), by monitoring physical
manifestations (e.g., inflammation, tumor size, etc.), or by an
accepted clinical assessment scale, for example, the Expanded
Disability Status Scale (for multiple sclerosis), the Irvine
Inflammatory Bowel Disease Questionnaire (32 point assessment
evaluates quality of life with respect to bowel function, systemic
symptoms, social function and emotional status--score ranges from
32 to 224, with higher scores indicating a better quality of life),
the Quality of Life Rheumatoid Arthritis Scale, or other accepted
clinical assessment scale as known in the field. A sustained (e.g.,
one day or more, preferably longer) reduction in disease or
disorder symptoms by at least 10% or by one or more points on a
given clinical scale is indicative of "effective" treatment.
Similarly, prophylaxis performed using a composition as described
herein is "effective" if the onset or severity of one or more
symptoms is delayed, reduced or abolished relative to such symptoms
in a similar individual (human or animal model) not treated with
the composition.
[0383] A composition containing a ligand or cocktail thereof
according to the present invention may be utilised in prophylactic
and therapeutic settings to aid in the alteration, inactivation,
killing or removal of a select target cell population in a mammal.
In addition, the selected repertoires of polypeptides described
herein may be used extracorporeally or in vitro selectively to
kill, deplete or otherwise effectively remove a target cell
population from a heterogeneous collection of cells. Blood from a
mammal may be combined extracorporeally with the ligands, e.g.
antibodies, cell-surface receptors or binding proteins thereof
whereby the undesired cells are killed or otherwise removed from
the blood for return to the mammal in accordance with standard
techniques.
[0384] I: Use of Half-Life Enhanced Dual-Specific Ligands According
to the Invention
[0385] Dual-specific ligands according to the method of the present
invention may be employed in in vivo therapeutic and prophylactic
applications, in vivo diagnostic applications and the like.
[0386] Therapeutic and prophylactic uses of dual-specific ligands
prepared according to the invention involve the administration of
ligands according to the invention to a recipient mammal, such as a
human. Dual specific antibodies according to the invention comprise
at least one specificity for a half-life enhancing molecule; one or
more further specificities may be directed against target
molecules. For example, a dual-specific IgG may be specific for
four epitopes, one of which is on a half-life enhancing molecule.
Dual-specificity can allow antibodies to bind to multimeric antigen
with great avidity. Dual-specific antibodies can allow the
cross-linking of two antigens, for example in recruiting cytotoxic
T-cells to mediate the killing of tumour cell lines.
[0387] Substantially pure ligands or binding proteins thereof, such
as dAb monomers, of at least 90 to 95% homogeneity are preferred
for administration to a mammal, and 98 to 99% or more homogeneity
is most preferred for pharmaceutical uses, especially when the
mammal is a human. Once purified, partially or to homogeneity as
desired, the ligands may be used diagnostically or therapeutically
(including extracorporeally) or in developing and performing assay
procedures, immunofluorescent stainings and the like (Lefkovite and
Pernis, (1979 and 1981) Immunological Methods, Volumes I and II,
Academic Press, NY).
[0388] The ligands of the present invention will typically find use
in preventing, suppressing or treating inflammatory states,
allergic hypersensitivity, cancer, bacterial or viral infection,
and autoimmune disorders (which include, but are not limited to,
Type I diabetes, multiple sclerosis, rheumatoid arthritis, systemic
lupus erythematosus, Crohn's disease and myasthenia gravis).
[0389] In the instant application, the term "prevention" involves
administration of the protective composition prior to the induction
of the disease. "Suppression" refers to administration of the
composition after an inductive event, but prior to the clinical
appearance of the disease. "Treatment" involves administration of
the protective composition after disease symptoms become
manifest.
[0390] Animal model systems which can be used to screen the
effectiveness of the dual specific ligands in protecting against or
treating the disease are available. Methods for the testing of
systemic lupus erythematosus (SLE) in susceptible mice are known in
the art (Knight et al. (1978) J. Exp. Med., 147: 1653; Reinersten
et al. (1978) New Eng. J. Med., 299: 515). Myasthenia Gravis (MG)
is tested in SJL/J female mice by inducing the disease with soluble
AchR protein from another species (Lindstrom et al. (1988) Adv.
Immunol., 42: 233). Arthritis is induced in a susceptible strain of
mice by injection of Type II collagen (Stuart et al. (1984) Ann.
Rev. Immunol., 42: 233). A model by which adjuvant arthritis is
induced in susceptible rats by injection of mycobacterial heat
shock protein has been described (Van Eden et al. (1988) Nature,
331: 171). Thyroiditis is induced in mice by administration of
thyroglobulin as described (Maron et al. (1980) J. Exp. Med., 152:
1115). Insulin dependent diabetes mellitus (IDDM) occurs naturally
or can be induced in certain strains of mice such as those
described by Kanasawa et al. (1984) Diabetologia, 27: 113. EAE in
mouse and rat serves as a model for MS in human. In this model, the
demyelinating disease is induced by administration of myelin basic
protein (see Paterson (1986) Textbook of Immunopathology, Mischer
et al., eds., Grune and Stratton, New York, pp. 179-213; McFarlin
et al. (1973) Science, 179: 478: and Satoh et al. (1987) J.
Immunol., 138: 179).
[0391] Dual specific ligands according to the invention and dAb
monomers able to bind to extracellular targets involved in
endocytosis (e.g. Clathrin) enable dual specific ligands to be
endocytosed, enabling another specificity able to bind to an
intracellular target to be delivered to an intracellular
environment. This strategy requires a dual specific ligand with
physical properties that enable it to remain functional inside the
cell. Alternatively, if the final destination intracellular
compartment is oxidising, a well folding ligand may not need to be
disulphide free.
[0392] Generally, the present dual specific ligands will be
utilised in purified form together with pharmacologically
appropriate carriers. Typically, these carriers include aqueous or
alcoholic/aqueous solutions, emulsions or suspensions, any
including saline and/or buffered media. Parenteral vehicles include
sodium chloride solution, Ringer's dextrose, dextrose and sodium
chloride and lactated Ringer's. Suitable physiologically-acceptable
adjuvants, if necessary to keep a polypeptide complex in
suspension, may be chosen from thickeners such as
carboxymethylcellulose, polyvinylpyrrolidone, gelatin and
alginates.
[0393] Intravenous vehicles include fluid and nutrient replenishers
and electrolyte replenishers, such as those based on Ringer's
dextrose. Preservatives and other additives, such as
antimicrobials, antioxidants, chelating agents and inert gases, may
also be present (Mack (1982) Remington's Pharmaceutical Sciences,
16th Edition).
[0394] The ligands of the present invention may be used as
separately administered compositions or in conjunction with other
agents. These can include various immunotherapeutic drugs, such as
cylcosporine, methotrexate, adriamycin or cisplatinum, and
immunotoxins. Pharmaceutical compositions can include "cocktails"
of various cytotoxic or other agents in conjunction with the
ligands of the present invention.
[0395] The route of administration of pharmaceutical compositions
according to the invention may be any of those commonly known to
those of ordinary skill in the art. For therapy, including without
limitation immunotherapy, the ligands of the invention can be
administered to any patient in accordance with standard techniques.
The administration can be by any appropriate mode, including
parenterally, intravenously, intramuscularly, intraperitoneally,
transdermally, via the pulmonary route, or also, appropriately, by
direct infusion with a catheter. The dosage and frequency of
administration will depend on the age, sex and condition of the
patient, concurrent administration of other drugs,
counterindications and other parameters to be taken into account by
the clinician.
[0396] The ligands of the invention can be lyophilised for storage
and reconstituted in a suitable carrier prior to use. This
technique has been shown to be effective with conventional
immunoglobulins and art-known lyophilisation and reconstitution
techniques can be employed. It will be appreciated by those skilled
in the art that lyophilisation and reconstitution can lead to
varying degrees of antibody activity loss (e.g. with conventional
immunoglobulins, IgM antibodies tend to have greater activity loss
than IgG antibodies) and that use levels may have to be adjusted
upward to compensate.
[0397] The compositions containing the present ligands or a
cocktail thereof can be administered for prophylactic and/or
therapeutic treatments. In certain therapeutic applications, an
adequate amount to accomplish at least partial inhibition,
suppression, modulation, killing, or some other measurable
parameter, of a population of selected cells is defined as a
"therapeutically-effective dose". Amounts needed to achieve this
dosage will depend upon the severity of the disease and the general
state of the patient's own immune system, but generally range from
0.005 to 5.0 mg of ligand per kilogram of body weight, with doses
of 0.05 to 2.0 mg/kg/dose being more commonly used. For
prophylactic applications, compositions containing the present
ligands or cocktails thereof may also be administered in similar or
slightly lower dosages.
[0398] A composition containing a ligand according to the present
invention may be utilised in prophylactic and therapeutic settings
to aid in the alteration, inactivation, killing or removal of a
select target cell population in a mammal.
[0399] In addition, the selected repertoires of polypeptides
described herein may be used extracorporeally or in vitro
selectively to kill, deplete or otherwise effectively remove a
target cell population from a heterogeneous collection of cells.
Blood from a mammal may be combined extracorporeally with the
ligands, e.g. antibodies, cell-surface receptors or binding
proteins thereof whereby the undesired cells are killed or
otherwise removed from the blood for return to the mammal in
accordance with standard techniques.
[0400] The invention is further described, for the purposes of
illustration only, in the following examples. As used herein, for
the purposes of dAb nomenclature, human TNF.alpha. is referred to
as TAR1 and human TNF.alpha. receptor 1 (p55 receptor) is referred
to as TAR2.
EXAMPLE 1
Selection of a Dual Specific scFv Antibody (K8) Directed Against
Human Serum Albumin (HSA) and .beta.-galactosidase (.beta.-gal)
[0401] This example explains a method for making a dual specific
antibody directed against .beta.-gal and HSA in which a repertoire
of V.sub..kappa. variable domains linked to a germline (dummy)
V.sub.H domain is selected for binding to .beta.-gal and a
repertoire of V.sub.H variable domains linked to a germline (dummy)
V.sub..kappa. domain is selected for binding to HSA. The selected
variable V.sub.H HSA and V.sub..kappa. .beta.-gal domains are then
combined and the antibodies selected for binding to .beta.-gal and
HSA. HSA is a half-life increasing protein found in human
blood.
[0402] Four human phage antibody libraries were used in this
experiment. TABLE-US-00001 Library 1 Germline V.sub..kappa./DVT
V.sub.H 8.46 .times. 10.sup.7 Library 2 Germline V.sub..kappa./NNK
V.sub.H 9.64 .times. 10.sup.7 Library 3 Germline V.sub.H/DVT
V.sub..kappa. 1.47 .times. 10.sup.8 Library 4 Germline V.sub.H/NNK
V.sub..kappa. 1.45 .times. 10.sup.8
[0403] All libraries are based on a single human framework for
V.sub.H (V3-23/DP47 and J.sub.H4b) and V.sub..kappa. (O12/O2/DPK9
and J.sub..kappa.1) with side chain diversity incorporated in
complementarity determining regions (CDR2 and CDR3).
[0404] Library 1 and Library 2 contain a dummy V.sub..kappa.
sequence, whereas the sequence of V.sub.H is diversified at
positions H50, H52, H52a, H53, H55, H56, H58, H95, H96, H97 and H98
(DVT or NNK encoded, respectively) (FIG. 1). Library 3 and Library
4 contain a dummy V.sub.H sequence, whereas the sequence of
V.sub..kappa. is diversified at positions L50, L53, L91, L92, L93,
L94 and L96 (DVT or NNK encoded, respectively) (FIG. 1). The
libraries are in phagemid pIT2/ScFv format (FIG. 2) and have been
preselected for binding to generic ligands, Protein A and Protein
L, so that the majority of clones in the unselected libraries are
functional. The sizes of the libraries shown above correspond to
the sizes after preselection. Library 1 and Library 2 were mixed
prior to selections on antigen to yield a single V.sub.H/dummy
V.sub..kappa. library and Library 3 and Library 4 were mixed to
form a single V.sub..kappa./dummy V.sub.H library.
[0405] Three rounds of selections were performed on .beta.-gal
using V.sub..kappa./dummy V.sub.H library and three rounds of
selections were performed on HSA using V.sub.H/dummy V.sub..kappa.
library. In the case of .beta.-gal the phage titres went up from
1.1.times.10.sup.6 in the first round to 2.0.times.10.sup.8 in the
third round. In the case of HSA the phage titres went up from
2.times.10.sup.4 in the first round to 1.4.times.10.sup.9 in the
third round. The selections were performed as described by Griffith
et al., (1993), except that KM13 helper phage (which contains a
pIII protein with a protease cleavage site between the D2 and D3
domains) was used and phage were eluted with 1 mg/ml trypsin in
PBS. The addition of trypsin cleaves the pIII proteins derived from
the helper phage (but not those from the phagemid) and elutes bound
scFv-phage fusions by cleavage in the c-myc tag (FIG. 2), thereby
providing a further enrichment for phages expressing functional
scFvs and a corresponding reduction in background (Kristensen &
Winter, Folding & Design 3: 321-328, Jul. 9, 1998). Selections
were performed using immunotubes coated with either HSA or
.beta.-gal at 100 .mu.g/ml concentration.
[0406] To check for binding, 24 colonies from the third round of
each selection were screened by monoclonal phage ELISA. Phage
particles were produced as described by Harrison et al., Methods
Enzymol. 1996;267:83-109. 96-well ELISA plates were coated with 100
.mu.l of HSA or .beta.-gal at 10 .mu.g/ml concentration in PBS
overnight at 4.degree. C. A standard ELISA protocol was followed
(Hoogenboom et al., 1991) using detection of bound phage with
anti-M13-HRP conjugate. A selection of clones gave ELISA signals of
greater than 1.0 with 50 .mu.l supernatant.
[0407] Next, DNA preps were made from V.sub.H/dummy V.sub..kappa.
library selected on HSA and from V.sub..kappa./dummy V.sub.H
library selected on .beta.-gal using the QIAprep Spin Miniprep kit
(Qiagen). To access most of the diversity, DNA preps were made from
each of the three rounds of selections and then pulled together for
each of the antigens. DNA preps were then digested with SalI/NotI
overnight at 37.degree. C. Following gel purification of the
fragments, V.sub..kappa. chains from the V.sub..kappa./dummy
V.sub.H library selected on .beta.-gal were ligated in place of a
dummy V.sub..kappa. chain of the V.sub.H/dummy V.sub..kappa.
library selected on HSA creating a library of 3.3.times.10.sup.9
clones.
[0408] This library was then either selected on HSA (first round)
and .beta.-gal (second round), HSA/.beta.-gal selection, or on
.beta.-gal (first round) and HSA (second round), .beta.-gal/HSA
selection. Selections were performed as described above. In each
case after the second round 48 clones were tested for binding to
HSA and .beta.-gal by the monoclonal phage ELISA (as described
above) and by ELISA of the soluble scFv fragments. Soluble antibody
fragments were produced as described by Harrison et al., (1996),
and standard ELISA protocol was followed Hoogenboom et al. (1991)
Nucleic Acids Res., 19: 4133, except that 2% Tween/PBS was used as
a blocking buffer and bound scFvs were detected with Protein L-HRP.
Three clones (E4, E5 and E8) from the HSA/.beta.-gal selection and
two clones (K8 and K10) from the .beta.-gal/HSA selection were able
to bind both antigens. scFvs from these clones were PCR amplified
and sequenced as described by Ignatovich et al., (1999) J Mol Biol
Nov. 26, 1999;294(2):457-65, using the primers LMB3 and pHENseq.
Sequence analysis revealed that all clones were identical.
Therefore, only one clone encoding a dual specific antibody (K8)
was chosen for further work (FIG. 3).
EXAMPLE 2
Characterisation of the Binding Properties of the K8 Antibody
[0409] Firstly, the binding properties of the K8 antibody were
characterised by the monoclonal phage ELISA. A 96-well plate was
coated with 100 .mu.l of HSA and .beta.-gal alongside with alkaline
phosphatase (APS), bovine serum albumin (BSA), peanut agglutinin,
lysozyme and cytochrome c (to check for cross-reactivity) at 10
.mu.g/ml concentration in PBS overnight at 4.degree. C. The
phagemid from K8 clone was rescued with KM13 as described by
Harrison et al., (1996) and the supernatant (50 .mu.l) containing
phage assayed directly. A standard ELISA protocol was followed
(Hoogenboom et al., 1991) using detection of bound phage with
anti-M13-HRP conjugate. The dual specific K8 antibody was found to
bind to HSA and .beta.-gal when displayed on the surface of the
phage with absorbance signals greater than 1.0 (FIG. 4). Strong
binding to BSA was also observed (FIG. 4). Since HSA and BSA are
76% homologous on the amino acid level, it is not surprising that
K8 antibody recognised both of these structurally related proteins.
No cross-reactivity with other proteins was detected (FIG. 4).
[0410] Secondly, the binding properties of the K8 antibody were
tested in a soluble scFv ELISA. Production of the soluble scFv
fragment was induced by IPTG as described by Harrison et al.,
(1996). To determine the expression levels of K8 scFv, the soluble
antibody fragments were purified from the supernatant of 50 ml
inductions using Protein A-Sepharose columns as described by Harlow
and Lane, Antibodies: a Laboratory Manual, (1988) Cold Spring
Harbor. OD.sub.280 was then measured and the protein concentration
calculated as described by Sambrook et al., (1989). K8 scFv was
produced in supernatant at 19 mg/l.
[0411] A soluble scFv ELISA was then performed using known
concentrations of the K8 antibody fragment. A 96-well plate was
coated with 100 .mu.l of HSA, BSA and .beta.-gal at 10 .mu.g/ml and
100 .mu.l of Protein A at 1 .mu.g/ml concentration. 50 .mu.l of the
serial dilutions of the K8 scFv was applied and the bound antibody
fragments were detected with Protein L-HRP. ELISA results confirmed
the dual specific nature of the K8 antibody (FIG. 5).
[0412] To confirm that binding to .beta.-gal is determined by the
V.sub..kappa. domain and binding to HSA/BSA by the V.sub.H domain
of the K8 scFv antibody, the V.sub..kappa. domain was cut out from
K8 scFv DNA by SalI/NotI digestion and ligated into a SalI/NotI
digested pIT2 vector containing dummy V.sub.H chain (FIGS. 1 and
2). Binding characteristics of the resulting clone
K8V.sub..kappa./dummy V.sub.H were analysed by soluble scFv ELISA.
Production of the soluble scFv fragments was induced by IPTG as
described by Harrison et al., (1996) and the supernatant (50.mu.)
containing scFvs assayed directly. Soluble scFv ELISA was performed
as described in Example 1 and the bound scFvs were detected with
Protein L-HRP. The ELISA results revealed that this clone was still
able to bind .beta.-gal, whereas binding to BSA was abolished (FIG.
6).
EXAMPLE 3
Selection of Single V.sub.H Domain Antibodies Antigens A and B and
Single V.sub..kappa. Domain Antibodies Directed Against Antigens C
and D
[0413] This example describes a method for making single V.sub.H
domain antibodies directed against antigens A and B and single
V.sub..kappa. domain antibodies directed against antigens C and D
by selecting repertoires of virgin single antibody variable domains
for binding to these antigens in the absence of the complementary
variable domains.
[0414] Selections and characterisation of the binding clones is
performed as described previously (see Example 5, PCT/GB
02/003014). Four clones are chosen for further work:
[0415] VH1-Anti A V.sub.H
[0416] VH2-Anti B V.sub.H
[0417] VK1-Anti C V.sub..kappa.
[0418] VK2-Anti D V.sub..kappa.
[0419] The procedures described above in Examples 1-3 may be used,
in a similar manner as that described, to produce dimer molecules
comprising combinations of V.sub.H domains (i.e., V.sub.H-V.sub.H
ligands) and cominations of V.sub.L domains (V.sub.L-V.sub.L
ligands).
EXAMPLE 4
Creation and Characterisation of the Dual Specific ScFv Antibodies
(VH1/VH2 Directed Against Antigens A and B and VK1/VK2 Directed
Against Antigens C and D)
[0420] This example demonstrates that dual specific ScFv antibodies
(VH1/VH2 directed against antigens A and B and VK1/VK2 directed
against antigens C and D) could be created by combining
V.sub..kappa. and V.sub.H single domains selected against
respective antigens in a ScFv vector.
[0421] To create dual specific antibody VH1/VH2, VH1 single domain
is excised from variable domain vector 1 (FIG. 7) by NcoI/XhoI
digestion and ligated into NcoI/XhoI digested variable domain
vector 2 (FIG. 7) to create VH1/variable domain vector 2. VH2
single domain is PCR amplified from variable domain vector 1 using
primers to introduce SalI restriction site to the 5' end and NotI
restriction site to the 3' end. The PCR product is then digested
with SalI/NotI and ligated into SalI/NotI digested VH1/variable
domain vector 2 to create VH1/VH2/variable domain vector 2.
[0422] VK1/VK2/variable domain vector 2 is created in a similar
way. The dual specific nature of the produced VH1/VH2 ScFv and
VK1/VK2 ScFv is tested in a soluble ScFv ELISA as described
previously (see Example 6, PCT/GB 02/003014). Competition ELISA is
performed as described previously (see Example 8, PCT/GB
02/003014).
[0423] Possible outcomes:
[0424] VH1/VH2 ScFv is able to bind antigens A and B
simultaneously
[0425] VK1/VK2 ScFv is able to bind antigens C and D
simultaneously
[0426] VH1/VH2 ScFv binding is competitive (when bound to antigen
A, VH1/VH2 ScFv cannot bind to antigen B)
[0427] VK1/VK2 ScFv binding is competitive (when bound to antigen
C, VK1/VK2 ScFv cannot bind to antigen D)
EXAMPLE 5
Construction of Dual Specific VH1/VH2 Fab and VK1/VK2 Fab and
Analysis of their Binding Properties
[0428] To create VH1/VH2 Fab, VH1 single domain is ligated into
NcoI/XhoI digested CH vector (FIG. 8) to create VH1/CH and VH2
single domain is ligated into SalI/NotI digested CK vector (FIG. 9)
to create VH2/CK. Plasmid DNA from VH1/CH and VH2/CK is used to
co-transform competent E. coli cells as described previously (see
Example 8, PCT/GB02/003014).
[0429] The clone containing VH1/CH and VH2/CK plasmids is then
induced by IPTG to produce soluble VH1/VH2 Fab as described
previously (see Example 8, PCT/GB 02/003014).
[0430] VK1/VK2 Fab is produced in a similar way.
[0431] Binding properties of the produced Fabs are tested by
competition ELISA as described previously (see Example 8, PCT/GB
02/003014).
[0432] Possible outcomes:
[0433] VH1/VH2 Fab is able to bind antigens A and B
simultaneously
[0434] VK1/VK2 Fab is able to bind antigens C and D
simultaneously
[0435] VH1/VH2 Fab binding is competitive (when bound to antigen A,
VH1/VH2 Fab cannot bind to antigen B)
[0436] VK1/VK2 Fab binding is competitive (when bound to antigen C,
VK1/VK2 Fab cannot bind to antigen D)
EXAMPLE 6
Chelating dAb Dimers
[0437] Summary
[0438] VH and VK homo-dimers are created in a dAb-linker-dAb format
using flexible polypeptide linkers. Vectors were created in the dAb
linker-dAb format containing glycine-serine linkers of different
lengths 3U:(Gly.sub.4Ser).sub.3, 5U:(Gly.sub.4Ser).sub.5,
7U:(Gly.sub.4Ser).sub.7. Dimer libraries were created using guiding
dAbs upstream of the linker: TAR1-5 (VK), TAR1-27(VK), TAR2-5(VH)
or TAR2-6(VK) and a library of corresponding second dAbs after the
linker. Using this method, novel dimeric dAbs were selected. The
effect of dimerisation on antigen binding was determined by ELISA
and BIAcore studies and in cell neutralisation and receptor binding
assays. Dimerisation of both TAR1-5 and TAR1-27 resulted in
significant improvement in binding affinity and neutralisation
levels.
[0439] 1.0 Methods
[0440] 1.1 Library Generation
[0441] 1.1.1 Vectors
[0442] pEDA3U, pEDA5U and pEDA7U vectors were designed to introduce
different linker lengths compatible with the dAb-linker-dAb format.
For pEDA3U, sense and anti-sense 73-base pair oligo linkers were
annealed using a slow annealing program (95.degree. C.-5 mins,
80.degree. C.-10 mins, 70.degree. C.-15 mins, 56.degree. C.-15
mins, 42.degree. C. until use) in buffer containing 0.1MNaCl, 10 mM
Tris-HCl pH7.4 and cloned using the Xhol and Notl restriction
sites. The linkers encompassed 3 (Gly.sub.4Ser) units and a stuffer
region housed between Sall and Notl cloning sites (scheme 1). In
order to reduce the possibility of monomeric dAbs being selected
for by phage display, the stuffer region was designed to include 3
stop codons, a Sac1 restriction site and a frame shift mutation to
put the region out of frame when no second dAb was present. For
pEDA5U and 7U due to the length of the linkers required,
overlapping oligo-linkers were designed for each vector, annealed
and elongated using Klenow. The fragment was then purified and
digested using the appropriate enzymes before cloning using the
Xhol and Notl restriction sites. ##STR1##
[0443] 1.1.2 Library Preparation
[0444] The N-terminal V gene corresponding to the guiding dAb was
cloned upstream of the linker using Ncol and Xhol restriction
sites. VH genes have existing compatible sites, however cloning VK
genes required the introduction of suitable restriction sites. This
was achieved by using modifying PCR primers (VK-DLIBF: 5'
cggccatggcgtcaacggacat 3' (SEQ ID NO:208); VKXholR: 5'
atgtgcgctcgagcgtttgattt 3' (SEQ ID NO:209)) in 30 cycles of PCR
amplification using a 2:1 mixture of SuperTaq (HTBiotechnology Ltd)
and pfu turbo (Stratagene). This maintained the Ncol site at the 5'
end while destroying the adjacent Sall site and introduced the Xhol
site at the 3' end. 5 guiding dAbs were cloned into each of the 3
dimer vectors: TAR1-5 (VK), TAR1-27(VK), TAR2-5(VH), TAR2-6(VK) and
TAR2-7(VK). All constructs were verified by sequence analysis.
[0445] Having cloned the guiding dAbs upstream of the linker in
each of the vectors (pEDA3U, 5U and 7U): TAR1-5 (VK), TAR1-27(VK),
TAR2-5(VH) or TAR2-6(VK) a library of corresponding second dAbs
were cloned after the linker. To achieve this, the complimentary
dAb libraries were PCR amplified from phage recovered from round 1
selections of either a VK library against Human TNF.alpha. (at
approximately 1.times.10.sup.6 diversity after round 1) when TAR1-5
or TAR1-27 are the guiding dAbs, or a VH or VK library against
human p55 TNF receptor (both at approximately 1.times.10.sup.5
diversity after round 1) when TAR2-5 or TAR2-6 respectively are the
guiding dAbs. For VK libraries PCR amplification was conducted
using primers in 30 cycles of PCR amplification using a 2:1 mixture
of SuperTaq and pfu turbo. VH libraries were PCR amplified using
primers in order to introduce a Sall restriction site at the 5' end
of the gene. The dAb library PCRs were digested with the
appropriate restriction enzymes, ligated into the corresponding
vectors down stream of the linker, using Sall/Notl restriction
sites and electroporated into freshly prepared competent TG1
cells.
[0446] The titres achieved for each library are as follows:
[0447] TAR1-5: pEDA3U=4.times.10.sup.8, pEDA5U=8.times.10.sup.7,
pEDA7U=1.times.10.sup.8
[0448] TAR1-27: pEDA3U=6.2.times.10.sup.8, pEDA5U=1.times.10.sup.8,
pEDA7U=1.times.10.sup.9
[0449] TAR2h-5: pEDA3U=4.times.10.sup.7, pEDA5U=2.times.10.sup.8,
pEDA7U=8.times.10.sup.7
[0450] TAR2h-6: pEDA3U=7.4.times.10.sup.8,
pEDA5U=1.2.times.10.sup.8, pEDA7U=2.2.times.10.sup.8
[0451] 1.2 Selections
[0452] 1.2.1 TNF.alpha.
[0453] Selections were conducted using human TNF.alpha. passively
coated on immunotubes. Briefly, Immunotubes are coated overnight
with 1-4 mls of the required antigen. The immunotubes were then
washed 3 times with PBS and blocked with 2% milk powder in PBS for
1-2 hrs and washed a further 3 times with PBS. The phage solution
is diluted in 2% milk powder in PBS and incubated at room
temperature for 2 hrs. The tubes are then washed with PBS and the
phage eluted with 1 mg/ml trypsin-PBS. Three selection strategies
were investigated for the TAR1-5 dimer libraries. The first round
selections were carried out in immunotubes using human TNF.alpha.
coated at 1 .mu.g/ml or 20 .mu.g/ml with 20 washes in PBS 0.1%
Tween. TG1 cells are infected with the eluted phage and the titres
are determined (eg, Marks et al J Mol Biol. Dec. 5,
1991;222(3):581-97, Richmann et al Biochemistry. Aug. 31,
1993;32(34):8848-55).
[0454] The titres recovered were:
[0455] pEDA3U=2.8.times.10.sup.7 (1 .mu.g/ml TNF)
1.5.times.10.sup.8 (20 .mu.g/ml TNF),
[0456] pEDA5U=1.8.times.10.sup.7 (1 .mu.g/ml TNF),
1.6.times.10.sup.8 (20 .mu.g/ml TNF)
[0457] pEDA7U=8.times.10.sup.6 (1 .mu.g/ml TNF), 7.times.10.sup.7
(20 .mu.g/ml TNF).
[0458] The second round selections were carried out using 3
different methods. [0459] 1. In immunotubes, 20 washes with
overnight incubation followed by a further 10 washes. [0460] 2. In
immunotubes, 20 washes followed by 1 hr incubation at RT in wash
buffer with (1 .mu.g/ml TNF.alpha.) and 10 further washes.
[0461] 3. Selection on streptavidin beads using 33 pmoles
biotinylated human TNF.alpha. (Henderikx et al., 2002, Selection of
antibodies against biotinylated antigens. Antibody Phage Display:
Methods and protocols, Ed. O'Brien and Atkin, Humana Press). Single
clones from round 2 selections were picked into 96 well plates and
crude supernatant preps were made in 2 ml 96 well plate format.
TABLE-US-00002 Round 1 Human TNF.alpha.immunotube Round 2 Round 2
Round 2 coating selection selection selection concentration method
1 method 2 method 3 pEDA3U 1 .mu.g/ml 1 .times. 10.sup.9 1.8
.times. 10.sup.9 2.4 .times. 10.sup.10 pEDA3U 20 .mu.g/ml 6 .times.
10.sup.9 1.8 .times. 10.sup.10 8.5 .times. 10.sup.10 pEDA5U 1
.mu.g/ml 9 .times. 10.sup.8 1.4 .times. 10.sup.9 2.8 .times.
10.sup.10 pEDA5U 20 .mu.g/ml 9.5 .times. 10.sup.9 8.5 .times.
10.sup.9 2.8 .times. 10.sup.10 pEDA7U 1 .mu.g/ml 7.8 .times.
10.sup.8 1.6 .times. 10.sup.8 4 .times. 10.sup.10 pEDA7U 20
.mu.g/ml 1 .times. 10.sup.10 8 .times. 10.sup.9 1.5 .times.
10.sup.10
[0462] For TAR1-27, selections were carried out as described
previously with the following modifications. The first round
selections were carried out in immunotubes using human TNF.alpha.
coated at 1 .mu.g/ml or 20 .mu.g/ml with 20 washes in PBS 0.1%
Tween. The second round selections were carried out in immunotubes
using 20 washes with overnight incubation followed by a further 20
washes. Single clones from round 2 selections were picked into 96
well plates and crude supernatant preps were made in 2 ml 96 well
plate format.
[0463] TAR1-27 titres are as follows: TABLE-US-00003 Human
TNF.alpha.immunotube coating conc Round 1 Round 2 pEDA3U 1 .mu.g/ml
4 .times. 10.sup.9 6 .times. 10.sup.9 pEDA3U 20 .mu.g/ml 5 .times.
10.sup.9 4.4 .times. 10.sup.10 pEDA5U 1 .mu.g/ml 1.5 .times.
10.sup.9 1.9 .times. 10.sup.10 pEDA5U 20 .mu.g/ml 3.4 .times.
10.sup.9 3.5 .times. 10.sup.10 pEDA7U 1 .mu.g/ml 2.6 .times.
10.sup.9 5 .times. 10.sup.9 pEDA7U 20 .mu.g/ml 7 .times. 10.sup.9
1.4 .times. 10.sup.10
[0464] 1.2.2 TNF Receptor 1 (p55 Receptor; TAR2)
[0465] Selections were conducted as described previously for the
TAR2h-5 libraries only. 3 rounds of selections were carried out in
immunotubes using either 1 .mu.g/ml human p55 TNF receptor or 10
.mu.g/ml human p55 TNF receptor with 20 washes in PBS 0.1% Tween
with overnight incubation followed by a further 20 washes. Single
clones from round 2 and 3 selections were picked into 96 well
plates and crude supernatant preps were made in 2 ml 96 well plate
format.
[0466] TAR2h-5 titres are as follows: TABLE-US-00004 Round 1 human
p55 TNF receptor immunotube coating concentration Round 1 Round 2
Round 3 pEDA3U 1 .mu.g/ml 2.4 .times. 10.sup.6 1.2 .times. 10.sup.7
1.9 .times. 10.sup.9 pEDA3U 10 .mu.g/ml 3.1 .times. 10.sup.7 7
.times. 10.sup.7 1 .times. 10.sup.9 pEDA5U 1 .mu.g/ml 2.5 .times.
10.sup.6 1.1 .times. 10.sup.7 5.7 .times. 10.sup.8 pEDA5U 10
.mu.g/ml 3.7 .times. 10.sup.7 2.3 .times. 10.sup.8 2.9 .times.
10.sup.9 pEDA7U 1 .mu.g/ml 1.3 .times. 10.sup.6 1.3 .times.
10.sup.7 1.4 .times. 10.sup.9 pEDA7U 10 .mu.g/ml 1.6 .times.
10.sup.7 1.9 .times. 10.sup.7 3 .times. 10.sup.10
[0467] 1.3 Screening
[0468] Single clones from round 2 or 3 selections were picked from
each of the 3U, 5U and 7U libraries from the different selections
methods, where appropriate. Clones were grown in 2.times. TY with
100 .mu.g/ml ampicillin and 1% glucose overnight at 37.degree. C. A
1/100 dilution of this culture was inoculated into 2 mls of
2.times. TY with 100 .mu.g/ml ampicillin and 0.1% glucose in 2 ml,
96 well plate format and grown at 37.degree. C. shaking until OD600
was approximately 0.9. The culture was then induced with 1 mM IPTG
overnight at 30.degree. C. The supernatants were clarified by
centrifugation at 400 rpm for 15 mins in a sorval plate centrifuge.
The supernatant preps the used for initial screening.
[0469] 1.3.1 ELISA
[0470] Binding activity of dimeric recombinant proteins was
compared to monomer by Protein A/L ELISA or by antigen ELISA.
Briefly, a 96 well plate is coated with antigen or Protein A/L
overnight at 4.degree. C. The plate washed with 0.05% Tween-PBS,
blocked for 2 hrs with 2% Tween-PBS. The sample is added to the
plate incubated for 1 hr at room temperature. The plate is washed
and incubated with the secondary reagent for 1 hr at room
temperature. The plate is washed and developed with TMB substrate.
Protein A/L-HRP or India-HRP was used as a secondary reagent. For
antigen ELISAs, the antigen concentrations used were 1 .mu.g/ml in
PBS for Human TNF.alpha. and human THF receptor 1. Due to the
presence of the guiding dAb in most cases dimers gave a positive
ELISA signal therefore off rate determination was examined by
BIAcore.
[0471] 1.3.2 BIAcore
[0472] BIAcore analysys was conducted for TAR1-5 and TAR2h-5
clones. For screening, Human TNF.alpha. was coupled to a CM5 chip
at high density (approximately 10000 RUs). 50 .mu.l of Human
TNF.alpha. (50 .mu.g/ml) was coupled to the chip at 5 .mu./min in
acetate buffer--pH5.5. Regeneration of the chip following analysis
using the standard methods is not possible due to the instability
of Human TNF.alpha., therefore after each sample was analysed, the
chip was washed for 10 mins with buffer.
[0473] For TAR1-5, clones supernatants from the round 2 selection
were screened by BIAcore.
[0474] 48 clones were screened from each of the 3U, 5U and 7U
libraries obtained using the following selection methods:
[0475] R1: 1 .mu.g/ml human TNF.alpha. immunotube, R2 1 .mu.g/ml
human TNF.alpha. immunotube, overnight wash.
[0476] R1: 20 .mu.g/ml human TNF.alpha. immunotube, R2 20 .mu.g/ml
human TNF.alpha. immunotube, overnight wash.
[0477] R1: 1 .mu.g/ml human TNF.alpha. immunotube, R2 33 pmoles
biotinylated human TNF.alpha. on beads.
[0478] R1: 20 .mu.g/ml human TNF.alpha. immunotube, R2 33 pmoles
biotinylated human TNF.alpha. beads.
[0479] For screening, human p55 TNF receptor was coupled to a CM5
chip at high density (approximately 4000 RUs). 100 .mu.l of human
p55 TNF receptor (10 .mu.g/ml) was coupled to the chip at 5
.mu.l/min in acetate buffer--pH5.5. Standard regeneration
conditions were examined (glycine pH2 or pH3) but in each case
antigen was removed from the surface of the chip therefore as with
TNF.alpha., therefore after each sample was analysed, the chip was
washed for 10 mins with buffer.
[0480] For TAR2-5, clones supernatants from the round 2 selection
were screened.
[0481] 48 clones were screened from each of the 3U, 5U and 7U
libraries, using the following selection methods:
[0482] R1: 1 .mu.g/ml human p55 TNF receptor immunotube, R2 1
.mu.g/ml human p55 TNF receptor immunotube, overnight wash.
[0483] R1: 10 .mu.g/ml human p55 TNF receptor immunotube, R2 10
.mu.g/ml human p55 TNF receptor immunotube, overnight wash.
[0484] 1.3.3 Receptor and Cell Assays
[0485] The ability of the dimers to neutralise in the receptor
assay was conducted as follows:
[0486] Receptor Binding
[0487] Anti-TNF dAbs were tested for the ability to inhibit the
binding of TNF to recombinant TNF receptor 1 (p55). Briefly,
Maxisorp plates were incubated overnight with 30 mg/ml anti-human
Fc mouse monoclonal antibody (Zymed, San Francisco, USA). The wells
were washed with phosphate buffered saline (PBS) containing 0.05%
Tween-20 and then blocked with 1% BSA in PBS before being incubated
with 100 ng/ml TNF receptor 1 Fc fusion protein (R&D Systems,
Minneapolis, USA). Anti-TNF dAb was mixed with TNF which was added
to the washed wells at a final concentration of 10 ng/ml. TNF
binding was detected with 0.2 mg/ml biotinylated anti-TNF antibody
(HyCult biotechnology, Uben, Netherlands) followed by 1 in 500
dilution of horse radish peroxidase labelled streptavidin (Amersham
Biosciences, UK) and then incubation with TMB substrate (KPL,
Gaithersburg, USA). The reaction was stopped by the addition of HCl
and the absorbance was read at 450 nm. Anti-TNF dAb activity lead
to a decrease in TNF binding and therefore a decrease in absorbance
compared with the TNF only control.
[0488] L929 Cytotoxicity Assay
[0489] Anti-TNF dAbs were also tested for the ability to neutralise
the cytotoxic activity of TNF on mouse L929 fibroblasts (Evans, T.
(2000) Molecular Biotechnology 15, 243-248). Briefly, L929 cells
plated in microtitre plates were incubated overnight with anti-TNF
dAb, 100 pg/ml TNF and 1 mg/ml actinomycin D (Sigma, Poole, UK).
Cell viability was measured by reading absorbance at 490 nm
following an incubation with
[3-(4,5-dimethylthiazol-2-yl)-5-(3-carbboxymethoxyphenyl)-2-(4-sulfopheny-
l)-2H-tetrazolium (Promega, Madison, USA). Anti-TNF dAb activity
lead to a decrease in TNF cytotoxicity and therefore an increase in
absorbance compared with the TNF only control.
[0490] In the initial screen, supernatants prepared for BIAcore
analysis, described above, were also used in the receptor assay.
Further analysis of selected dimers was also conducted in the
receptor and cell assays using purified proteins.
[0491] HeLa IL-8 Assay
[0492] Anti-TNFR1 or anti-TNF alpha dAbs were tested for the
ability to neutralise the induction of IL-8 secretion by TNF in
HeLa cells (method adapted from that of Akeson, L. et al (1996)
Journal of Biological Chemistry 271, 30517-30523, describing the
induction of IL-8 by IL-1 in HUVEC; here we look at induction by
human TNF alpha and we use HeLa cells instead of the HUVEC cell
line). Briefly, HeLa cells plated in microtitre plates were
incubated overnight with dAb and 300 pg/ml TNF. Post incubation the
supernatant was aspirated off the cells and IL-8 concentration
measured via a sandwich ELISA (R&D Systems). Anti-TNFR1 dAb
activity lead to a decrease in IL-8 secretion into the supernatant
compared with the TNF only control.
[0493] The L929 assay is used throughout the following experiments;
however, the use of the HeLa IL-8 assay is preferred to measure
anti-TNF receptor 1 (p55) ligands; the presence of mouse p55 in the
L929 assay poses certain limitations in its use.
[0494] 1.4 Sequence Analysis
[0495] Dimers that proved to have interesting properties in the
BIAcore and the receptor assay screens were sequenced. Sequences
are detailed in the sequence listing.
[0496] 1.5 Formatting
[0497] 1.5.1 TAR1-5-19 Dimers
[0498] The TAR1-5 dimers that were shown to have good
neutralisation properties were re-formatted and analysed in the
cell and receptor assays. The TAR1-5 guiding dab was substituted
with the affinity matured clone TAR1-5-19. To achieve this TAR1-5
was cloned out of the individual dimer pair and substituted with
TAR1-5-19 that had been amplified by PCR. In addition, TAR1-5-19
homodimers were also constructed in the 3U, 5U and 7U vectors. The
N terminal copy of the gene was amplified by PCR and cloned as
described above and the C-terminal gene fragment was cloned using
existing Sall and Notl restriction sites.
[0499] 1.5.2 Mutagenesis
[0500] The amber stop codon present in dAb2, one of the C-terminal
dAbs in the TAR1-5 dimer pairs was mutated to a glutamine by
site-directed mutagenesis.
[0501] 1.5.3 Fabs
[0502] The dimers containing TAR1-5 or TAR1-5-19 were re-formatted
into Fab expression vectors. dAbs were cloned into expression
vectors containing either the CK or CH genes using Sfil and Notl
restriction sites and verified by sequence analysis. The CK vector
is derived from a pUC based ampicillin resistant vector and the CH
vector is derived from a pACYC chloramphenicol resistant vector.
For Fab expression the dAb-CH and dAb-CK constructs were
co-transformed into HB2151 cells and grown in 2.times. TY
containing 0.1% glucose, 100 .mu.g/ml ampicillin and 10 .mu.g/ml
chloramphenicol.
[0503] 1.5.3 Hinge Dimerisation
[0504] Dimerisation of dAbs via cystine bond formation was
examined. A short sequence of amino acids EPKSGDKTHTCPPCP (SEQ ID
NO:210) a modified form of the human IgGC1 hinge was engineered at
the C terminal region on the dAb. An oligo linker encoding for this
sequence was synthesised and annealed, as described previously. The
linker was cloned into the pEDA vector containing TAR1-5-19 using
Xhol and Notl restriction sites. Dimerisation occurs in situ in the
periplasm.
[0505] 1.6 Expression and Purification
[0506] 1.6.1 Expression
[0507] Supernatants were prepared in the 2 ml, 96-well plate format
for the initial screening as described previously. Following the
initial screening process selected dimers were analysed further.
Dimer constructs were expressed in TOP10F' or HB2151 cells as
supernatants. Briefly, an individual colony from a freshly streaked
plate was grown overnight at 37.degree. C. in 2.times. TY with 100
.mu.g/ml ampicillin and 1% glucose. A 1/100 dilution of this
culture was inoculated into 2.times. TY with 100 .mu.g/ml
ampicillin and 0.1% glucose and grown at 37.degree. C. shaking
until OD600 was approximately 0.9. The culture was then induced
with 1 mM IPTG overnight at 30.degree. C. The cells were removed by
centrifugation and the supernatant purified with protein A or L
agarose.
[0508] Fab and cysteine hinge dimers were expressed as periplasmic
proteins in HB2152 cells. A 1/100 dilution of an overnight culture
was inoculated into 2.times. TY with 0.1% glucose and the
appropriate antibiotics and grown at 30.degree. C. shaking until
OD600 was approximately 0.9. The culture was then induced with 1 mM
IPTG for 3-4 hours at 25.degree. C. The cells were harvested by
centrifugation and the pellet resuspended in periplasmic
preparation buffer (30 mM Tris-HCl pH8.0, 1 mM EDTA, 20% sucrose).
Following centrifugation the supernatant was retained and the
pellet resuspended in 5 mM MgSO.sub.4. The supernatant was
harvested again by centrifugation, pooled and purified.
[0509] 1.6.2 Protein A/L Purification
[0510] Optimisation of the purification of dimer proteins from
Protein L agarose (Affitech, Norway) or Protein A agarose (Sigma,
UK) was examined. Protein was eluted by batch or by column elution
using a peristaltic pump. Three buffers were examined 0.1M
Phosphate-citrate buffer pH2.6, 0.2M Glycine pH2.5 and 0.1M Glycine
pH2.5. The optimal condition was determined to be under peristaltic
pump conditions using 0.1M Glycine pH2.5 over 10 column volumes.
Purification from protein A was conducted peristaltic pump
conditions using 0.1M Glycine pH2.5.
[0511] 1.6.3 FPLC Purification
[0512] Further purification was carried out by FPLC analysis on the
AKTA Explorer 100 system (Amersham Biosciences Ltd). TAR1-5 and
TAR1-5-19 dimers were fractionated by cation exchange
chromatography (1 ml Resource S--Amersham Biosciences Ltd) eluted
with a 0-1M NaCl gradient in 50 mM acetate buffer pH4. Hinge dimers
were purified by ion exchange (1 ml Resource Q Amersham Biosciences
Ltd) eluted with a 0-1M NaCl gradient in 25 mM Tris HCl pH 8.0.
Fabs were purified by size exclusion chromatography using a
superose 12 (Amersham Biosciences Ltd) column run at a flow rate of
0.5 ml/min in PBS with 0.05% tween. Following purification samples
were concentrated using vivaspin 5K cut off concentrators
(Vivascience Ltd).
[0513] 2.0 Results
[0514] 2.1 TAR1-5 Dimers
[0515] 6.times.96 clones were picked from the round 2 selection
encompassing all the libraries and selection conditions.
Supernatant preps were made and assayed by antigen and Protein L
ELISA, BIAcore and in the receptor assays. In ELISAs, positive
binding clones were identified from each selection method and were
distributed between 3U, 5U and 7U libraries. However, as the
guiding dAb is always present it was not possible to discriminate
between high and low affinity binders by this method therefore
BIAcore analysis was conducted.
[0516] BIAcore analysis was conducted using the 2 ml supernatants.
BIAcore analysis revealed that the dimer Koff rates were vastly
improved compared to monomeric TAR1-5. Monomer Koff rate was in the
range of 10.sup.-1M compared with dimer Koff rates which were in
the range of 10.sup.-3-10.sup.-4M. 16 clones that appeared to have
very slow off rates were selected, these came from the 3U, 5U and
7U libraries and were sequenced. In addition the supernatants were
analysed for the ability to neutralise human TNF.alpha. in the
receptor assay.
[0517] 6 lead clones (d1-d6 below) that neutralised in these assays
and have been sequenced. The results shows that out of the 6 clones
obtained there are only 3 different second dAbs (dAb1, dAb2 and
dAb3) however where the second dAb is found more than once they are
linked with different length linkers.
[0518] TAR1-5d1: 3U linker 2.sup.nd dAb=dAb1-1 .mu.g/ml Ag
immunotube overnight wash
[0519] TAR1-5d2: 3U linker 2.sup.nd dAb=dAb2-1 .mu.g/ml Ag
immunotube overnight wash
[0520] TAR1-5d3: 5U linker 2.sup.nd dAb=dAb2-1 .mu.g/ml Ag
immunotube overnight wash
[0521] TAR1-5d4: 5U linker 2.sup.nd dAb=dAb3-20 .mu.g/ml Ag
immunotube overnight wash
[0522] TAR1-5d5: 5U linker 2.sup.nd dAb=dAb1-20 .mu.g/ml Ag
immunotube overnight wash
[0523] TAR1-5d6: 7U linker 2.sup.nd dAb=dAb1-R1:1 .mu.g/ml Ag
immunotube overnight wash, R2:beads
[0524] The 6 lead clones were examined further. Protein was
produced from the periplasm and supernatant, purified with protein
L agarose and examined in the cell and receptor assays. The levels
of neutralisation were variable (Table 1). The optimal conditions
for protein preparation were determined. Protein produced from
HB2151 cells as supernatants gave the highest yield (approximately
10 mgs/L of culture). The supernatants were incubated with protein
L agarose for 2 hrs at room temperature or overnight at 4.degree.
C. The beads were washed with PBS/NaCl and packed onto an FPLC
column using a peristaltic pump. The beads were washed with 10
column volumes of PBS/NaCl and eluted with 0.1M glycine pH2.5. In
general, dimeric protein is eluted after the monomer.
[0525] TAR1-5d1-6 dimers were purified by FPLC. Three species were
obtained, by FPLC purification and were identified by SDS PAGE. One
species corresponds to monomer and the other two species
corresponds to dimers of different sizes. The larger of the two
species is possibly due to the presence of C terminal tags. These
proteins were examined in the receptor assay. The data presented in
table 1 represents the optimum results obtained from the two
dimeric species (FIG. 11)
[0526] The three second dAbs from the dimer pairs (ie, dAb1, dAb2
and dAb3) were cloned as monomers and examined by ELISA and in the
cell and receptor assay. All three dAbs bind specifically to TNF by
antigen ELISA and do not cross react with plastic or BSA. As
monomers, none of the dAbs neutralise in the cell or receptor
assays.
[0527] 2.1.2 TAR1-5-19 Dimers
[0528] TAR1-5-19 was substituted for TAR1-5 in the 6 lead clones.
Analysis of all TAR1-5-19 dimers in the cell and receptor assays
was conducted using total protein (protein L purified only) unless
otherwise stated (Table 2). TAR1-5-19d4 and TAR1-5-19d3 have the
best ND.sub.50 (.about.5 nM) in the cell assay, this is consistent
with the receptor assay results and is an improvement over
TAR1-5-19 monomer (ND.sub.50.about.30 nM). Although purified TAR1-5
dimers give variable results in the receptor and cell assays
TAR1-5-19 dimers were more consistent. Variability was shown when
using different elution buffers during the protein purification.
Elution using 0.1M Phosphate-citrate buffer pH2.6 or 0.2M Glycine
pH2.5 although removing all protein from the protein L agarose in
most cases rendered it less functional.
[0529] TAR1-5-19d4 was expressed in the fermenter and purified on
cation exchange FPLC to yield a completely pure dimer. As with
TAR1-5d4 three species were obtained, by FPLC purification
corresponding to monomer and two dimer species. This dimer was
amino acid sequenced. TAR1-5-19 monomer and TAR1-5-19d4 were then
examined in the receptor assay and the resulting IC50 for monomer
was 30 nM and for dimer was 8 nM. The results of the receptor assay
comparing TAR1-5-19 monomer, TAR1-5-19d4 and TAR1-5d4 is shown in
FIG. 10.
[0530] TAR1-5-19 homodimers were made in the 3U, 5U and 7U vectors,
expressed and purified on Protein L. The proteins were examined in
the cell and receptor assays and the resulting IC.sub.50s (for
receptor assay) and ND.sub.50s (for cell assay) were determined
(table 3, FIG. 12).
[0531] 2.2 Fabs
[0532] TAR1-5 and TAR1-5-19 dimers were also cloned into Fab
format, expressed and purified on protein L agarose. Fabs were
assessed in the receptor assays (Table 4). The results showed that
for both TAR1-5-19 and TAR1-5 dimers the neutralisation levels were
similar to the original Gly.sub.4Ser linker dimers from which they
were derived. A TAR1-5-19 Fab where TAR1-5-19 was displayed on both
CH and CK was expressed, protein L purified and assessed in the
receptor assay. The resulting IC50 was approximately 1 nM.
[0533] 2.3 TAR1-27 Dimers
[0534] 3.times.96 clones were picked from the round 2 selection
encompassing all the libraries and selection conditions. 2 ml
supernatant preps were made for analysis in ELISA and bioassays.
Antigen ELISA gave 71 positive clones. The receptor assay of crude
supernatants yielded 42 clones with inhibitory properties (TNF
binding 0-60%). In the majority of cases inhibitory properties
correlated with a strong ELISA signal. 42 clones were sequenced, 39
of these have unique second dAb sequences. The 12 dimers that gave
the best inhibitory properties were analysed further.
[0535] The 12 neutralising clones were expressed as 200 ml
supernatant preps and purified on protein L. These were assessed by
protein L and antigen ELISA, BIAcore and in the receptor assay.
Strong positive ELISA signals were obtained in all cases. BIAcore
analysis revealed all clones to have fast on and off rates. The off
rates were improved compared to monomeric TAR1-27, however the off
rate of TAR1-27 dimers was faster (Koff is approximately in the
range of 10.sup.-1 and 10.sup.-2M) than the TAR1-5 dimers examined
previously (Koff is approximately in the range of
10.sup.-3-10.sup.-4M). The stability of the purified dimers was
questioned and therefore in order to improve stability, the
addition on 5% glycerol, 0.5% Triton X100 or 0.5% NP40 (Sigma) was
included in the purification of 2 TAR1-27 dimers (d2 and d16).
Addition of NP40 or Triton X100.TM. improved the yield of purified
product approximately 2 fold. Both dimers were assessed in the
receptor assay. TAR1-27d2 gave IC50 of .about.30 nM under all
purification conditions. TAR1-27d16 showed no neutralisation effect
when purified without the use of stabilising agents but gave an
IC50 of .about.50 nM when purified under stabilising conditions. No
further analysis was conducted.
[0536] 2.4 TAR2-5 Dimers
[0537] 3.times.96 clones were picked from the second round
selections encompassing all the libraries and selection conditions.
2 ml supernatant preps were made for analysis. Protein A and
antigen ELISAs were conducted for each plate. 30 interesting clones
were identified as having good off-rates by BIAcore (Koff ranges
between 10.sup.-2-10.sup.-3M). The clones were sequenced and 13
unique dimers were identified by sequence analysis. TABLE-US-00005
TABLE 1 TAR1-5 dimers Cell Protein Elution Receptor/ Dimer type
Purification Fraction conditions Cell assay TAR1-5d1 HB2151 Protein
L + FPLC small dimeric 0.1 M glycine RA.about.30 nM species pH2.5
TAR1-5d2 HB2151 Protein L + FPLC small dimeric 0.1 M glycine
RA.about.50 nM species pH2.5 TAR1-5d3 HB2151 Protein L + FPLC large
dimeric 0.1 M glycine RA.about.300 nM species pH2.5 TAR1-5d4 HB2151
Protein L + FPLC small dimeric 0.1 M glycine RA.about.3 nM species
pH2.5 TAR1-5d5 HB2151 Protein L + FPLC large dimeric 0.1 M glycine
RA.about.200 nM species pH2.5 TAR1-5d6 HB2151 Protein L + FPLC
Large dimeric 0.1 M glycine RA.about.100 nM species pH2.5
[0538] note dimer 2 and dimer 3 have the same second dAb (called
dAb2), however have different linker lengths
(d2=(Gly.sub.4Ser).sub.3, d3=(Gly.sub.4Ser).sub.3). dAb1 is the
partner dAb to dimers 1, 5 and 6. dAb3 is the partner dAb to
dimer4. None of the partner dAbs neutralise alone. FPLC
purification is by cation exchange unless otherwise stated. The
optimal dimeric species for each dimer obtained by FPLC was
determined in these assays. TABLE-US-00006 TABLE 2 TAR1-5-19 dimers
Cell Protein Elution Receptor/Cell Dimer type Purification Fraction
conditions assay TAR1-5-19 d1 TOP10F' Protein L Total protein 0.1M
glycine pH 2.0 RA.about.15 nM TAR1-5-19 d2 TOP10F' Protein L Total
protein 0.1M glycine pH 2.0 + 0.05% RA.about.2 nM (no stop codon)
NP40 TAR1-5-19 d3 TOP10F' Protein L Total protein 0.1M glycine pH
2.5 + 0.05% RA.about.8 nM (no stop codon) NP40 TAR1-5-19 d4 TOP10F'
Protein L + FPLC FPLC purified 0.1M glycine RA.about.2-5 nM
fraction pH2.0 CA.about.12 nM TAR1-5-19 d5 TOP10F' Protein L Total
protein 0.1M glycine pH2.0 + NP40 RA.about.8 nM CA.about.10 nM
TAR1-5-19 d6 TOP10F' Protein L Total protein 0.1M glycine pH 2.0
RA.about.10 nM
[0539] TABLE-US-00007 TABLE 3 TAR1-5-19 homodimers Cell Protein
Elution Receptor/Cell Dimer type Purification Fraction conditions
assay TAR1-5-19 3U HB2151 Protein L Total protein 0.1M glycine
RA.about.20 nM homodimer pH2.5 CA.about.30 nM TAR1-5-19 5U HB2151
Protein L Total protein 0.1M glycine RA.about.2 nM homodimer pH2.5
CA.about.3 nM TAR1-5-19 7U HB2151 Protein L Total protein 0.1M
glycine RA.about.10 nM homodimer pH2.5 CA.about.15 nM TAR1-5-19 cys
HB2151 Protein L + FPLC FPLC purified 0.1M glycine RA.about.2 nM
hinge dimer fraction pH2.5 TAR1-5- HB2151 Protein Total protein
0.1M glycine RA.about.1 nM 19CH/TAR1- pH2.5 5-19 CK
[0540] TABLE-US-00008 TABLE 4 TAR1-5/TAR1-5-19 Fabs Cell Protein
Elution Receptor/Cell Dimer type Purification Fraction conditions
assay TAR1-5CH/ HB2151 Protein L Total protein 0.1M citrate
RA.about.90 nM dAb1 CK pH2.6 TAR1-5CH/ HB2151 Protein L Total
protein 0.1M glycine RA.about.30 nM dAb2 CK pH2.5 CA.about.60 nM
dAb3CH/ HB2151 Protein L Total protein 0.1M citrate RA.about.100 nM
TAR1-5CK pH2.6 TAR1-5- HB2151 Protein L Total protein 0.1M glycine
RA.about.6 nM 19CH/ pH2.0 dAb1 CK dAb1 CH/ HB2151 Protein L 0.1M
glycine Myc/flag RA.about.6 nM TAR1-5-19CK pH2.0 TAR1-5- HB2151
Protein L Total protein 0.1M glycine RA.about.8 nM 19CH/ pH2.0
CA.about.12 nM dAb2 CK TAR1-5- HB2151 Protein L Total protein 0.1M
glycine RA.about.3 nM 19CH/ pH2.0 dAb3CK
EXAMPLE 7
dAb Dimerisation by Terminal Cysteine Linkage
[0541] Summary
[0542] For dAb dimerisation, a free cysteine has been engineered at
the C-terminus of the protein. When expressed the protein forms a
dimer which can be purified by a two step purification method.
[0543] PCR Construction of TAR1-5-19CYS Dimer
[0544] See example 8 describing the dAb trimer. The trimer protocol
gives rise to a mixture of monomer, dimer and trimer.
[0545] Expression and Purification of TAR1-5-19CYS Dimer
[0546] The dimer was purified from the supernatant of the culture
by capture on Protein L agarose as outlined in the example 8.
[0547] Separation of TAR1-5-19CYS Monomer from the TAR1-5-19CYS
Dimer
[0548] Prior to cation exchange separation, the mixed monomer/dimer
sample was buffer exchanged into 50 mM sodium acetate buffer pH 4.0
using a PD-10 column (Amersham Pharmacia), following the
manufacturer's guidelines. The sample was then applied to a 1 mL
Resource S cation exchange column (Amersham Pharmacia), which had
been pre-equilibrated with 50 mM sodium acetate pH 4.0. The monomer
and dimer were separated using the following salt gradient in 50 mM
sodium acetate pH 4.0:
[0549] 150 to 200 mM sodium chloride over 15 column volumes
[0550] 200 to 450 mM sodium chloride over 10 column volumes
[0551] 450 to 1000 mM sodium chloride over 15 column volumes
[0552] Fractions containing dimer only were identified using
SDS-PAGE and then pooled and the pH increased to 8 by the addition
of 1/5 volume of 1M Tris pH 8.0.
[0553] In vitro Functional Binding Assay: TNF Receptor Assay and
Cell Assay
[0554] The affinity of the dimer for human TNF.alpha. was
determined using the TNF receptor and cell assay. IC50 in the
receptor assay was approximately 0.3-0.8 nM; ND50 in the cell assay
was approximately 3-8 nM.
[0555] Other Possible TAR1-5-19CYS Dimer Formats
[0556] PEG Dimers and Custom Synthetic Maleimide Dimers
[0557] Nektar (Shearwater) offer a range of bi-maleimide PEGs
[mPEG2-(MAL)2 or mPEG-(MAL)2] which would allow the monomer to be
formatted as a dimer, with a small linker separating the dAbs and
both being linked to a PEG ranging in size from 5 to 40 kDa. It has
been shown that the 5 kDa mPEG-(MAL)2 (ie,
[TAR1-5-19]-Cys-maleimide-PEG.times.2, wherein the maleimides are
linked together in the dimer) has an affinity in the TNF receptor
assay of .about.1-3 nM. Also the dimer can also be produced using
TMEA (Tris[2-maleimidoethyl]amine) (Pierce Biotechnology) or other
bi-functional linkers.
[0558] It is also possible to produce the disulphide dimer using a
chemical coupling procedure using 2,2'-dithiodipyridine (Sigma
Aldrich) and the reduced monomer.
[0559] Addition of a Polypeptide Linker or Hinge to the C-Terminus
of the dAb.
[0560] A small linker, either (Gly.sub.4Ser).sub.n where n=1 to 10,
eg, 1, 2, 3, 4, 5, 6 or 7, an immunoglobulin (eg, IgG hinge region
or random peptide sequence (eg, selected from a library of random
peptide sequences) can be engineered between the dAb and the
terminal cysteine residue. This can then be used to make dimers as
outlined above.
EXAMPLE 8
dAb Trimerisation
[0561] Summary
[0562] For dAb trimerisation, a free cysteine is required at the
C-terminus of the protein. The cysteine residue, once reduced to
give the free thiol, can then be used to specifically couple the
protein to a trimeric maleimide molecule, for example TMEA
(Tris[2-maleimidoethyl]amine).
[0563] PCR Construction of TAR1-5-19CYS
[0564] The following oligonucleotides were used to specifically PCR
TAR1-5-19 with a SalI and BamHI sites for cloning and also to
introduce a C-terminal cysteine residue: TABLE-US-00009 SalI
.about..about..about..about..about..about..about..about. Trp Ser
Ala Ser Thr Asp* Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala
Ser Val 1 TGG AGC GCG TCG ACG GAC ATC CAG ATG ACC CAG TCT CCA TCC
TCT CTG TCT GCA TCT GTA ACC TCG CGC AGC TGC CTG TAG GTC TAC TGG GTC
AGA GGT AGG AGA GAC AGA CGT AGA CAT Gly Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser Gln Ser Ile Asp Ser Tyr Leu His Trp 61 GGA GAC CGT GTC
ACC ATC ACT TGC CGG GCA AGT CAG AGC ATT GAT AGT TAT TTA CAT TGG CCT
CTG GCA CAG TGG TAG TGA ACG GCC CGT TCA GTC TCG TAA CTA TCA ATA AAT
GTA ACC Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser
Ala Ser Glu Leu Gln 121 TAC CAG CAG AAA CCA GGG AAA GCC CCT AAG CTC
CTG ATC TAT AGT GCA TCC GAG TTG CAA ATG GTC GTC TTT GGT CCC TTT CGG
GGA TTC GAG GAC TAG ATA TCA CGT AGG CTC AAC GTT Ser Gly Val Pro Ser
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 181 AGT
GGG GTC CCA TCA CGT TTC AGT GGC AGT GGA TCT GGG ACA GAT TTC ACT CTC
ACC ATC TCA CCC CAG GGT AGT GCA AAG TCA CCG TCA CCT AGA CCC TGT CTA
AAG TGA GAG TGG TAG Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr
Cys Gln Gln Val Val Trp Arg Pro 241 AGC AGT CTG CAA CCT GAA GAT TTT
GCT ACG TAC TAC TGT CAA CAG GTT GTG TGG CGT CCT TCG TCA GAC GTT GGA
CTT CTA AAA CGA TGC ATG ATG ACA GTT GTC CAA CAC ACC GCA GGA BamHI
.about..about..about..about..about..about..about..about. Phe Thr
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Cys *** *** Gly Ser Gly
301 TTT ACG TTC GGC CAA GGG ACC AAG GTG GAA ATC AAA CGG TGC TAA TAA
GGA TCC GGC AAA TGC AAG CCG GTT CCC TGG TTC CAC CTT TAG TTT GCC ACG
ATT ATT CCT AGG CCG
[0565] (* start of TAR1-5-19CYS sequence TAR1-5-19CYS amino acid
sequence (SEQ ID NO:211; TAR1-5-19CYS nucleotide sequences (SEQ ID
NO:212, coding strand; SEQ ID NO:213, noncoding strand))
TABLE-US-00010 Forward primer 5'-TGGAGCGCGTCGACGGACATCCAGATGACCC
(SEQ ID NO:214) AGTCTCCA-3' Reverse primer
5'-TTAGCAGCCGGATCCTTATTAGCACCGTTTG (SEQ ID NO:215) ATTTCCAC-3'
[0566] The PCR reaction (50 .mu.L volume) was set up as follows:
200 .mu.M dNTPs, 0.4 .mu.M of each primer, 5 .mu.L of 10.times. Pfu
Turbo buffer (Stratagene), 100 ng of template plasmid (encoding
TAR1-5-19), 1 .mu.L of Pfu Turbo enzyme (Stratagene) and the volume
adjusted to 50 .mu.L using sterile water. The following PCR
conditions were used: initial denaturing step. 94.degree. C. for 2
mins, then 25 cycles of 94.degree. C. for 30 secs, 64.degree. C.
for 30 sec and 72.degree. C. for 30 sec. A final extension step was
also included of 72.degree. C. for 5 mins. The PCR product was
purified and digested with SalI and BamHI and ligated into the
vector which had also been cut with the same restriction enzymes.
Correct clones were verified by DNA sequencing.
[0567] Expression and Purification of TAR1-5-19CYS
[0568] TAR1-5-19CYS vector was transformed into BL21 (DE3) pLysS
chemically competent cells (Novagen) following the manufacturer's
protocol. Cells carrying the dAb plasmid were selected for using
100 .mu.g/mL carbenicillin and 37 .mu.g/mL chloramphenicol.
Cultures were set up in 2L baffled flasks containing 500 mL of
terrific broth (Sigma-Aldrich), 100 .mu.g/mL carbenicillin and 37
.mu.g/mL chloramphenicol. The cultures were grown at 30.degree. C.
at 200 rpm to an O.D.600 of 1-1.5 and then induced with 1 mM IPTG
(isopropyl-beta-D-thiogalactopyranoside, from Melford
Laboratories). The expression of the dAb was allowed to continue
for 12-16 hrs at 30.degree. C. It was found that most of the dAb
was present in the culture media. Therefore, the cells were
separated from the media by centrifugation (8,000.times.g for 30
mins), and the supernatant used to purify the dAb. Per litre of
supernatant, 30 mL of Protein L agarose (Affitech) was added and
the dAb allowed to batch bind with stirring for 2 hours. The resin
was then allowed to settle under gravity for a further hour before
the supernatant was siphoned off. The agarose was then packed into
a XK 50 column (Amersham Phamacia) and was washed with 10 column
volumes of PBS. The bound dAb was eluted with 100 mM glycine pH 2.0
and protein containing fractions were then neutralized by the
addition of 1/5 volume of 1 M Tris pH 8.0. Per litre of culture
supernatant 20 mg of pure protein was isolated, which contained a
50:50 ratio of monomer to dimer.
[0569] Trimerisation of TAR1-5-19CYS
[0570] 2.5 ml of 100 .mu.M TAR1-5-19CYS was reduce with 5 mM
dithiothreitol and left at room temperature for 20 minutes. The
sample was then buffer exchanged using a PD-10 column (Amersham
Pharmacia). The column had been pre-equilibrated with 5 mM EDTA, 50
mM sodium phosphate pH 6.5, and the sample applied and eluted
following the manufactures guidelines. The sample was placed on ice
until required. TMEA (Tris[2-maleimidoethyl]amine) was purchased
from Pierce Biotechnology. A 20 mM stock solution of TMEA was made
in 100% DMSO (dimethyl sulphoxide). It was found that a
concentration of TMEA greater than 3:1 (molar ratio of dAb:TMEA)
caused the rapid precipitation and cross-linking of the protein.
Also the rate of precipitation and cross-linking was greater as the
pH increased. Therefore using 100 .mu.M reduced TAR1-5-19CYS, 25
.mu.M TMEA was added to trimerise the protein and the reaction
allowed to proceed at room temperature for two hours. It was found
that the addition of additives such as glycerol or ethylene glycol
to 20% (v/v), significantly reduced the precipitation of the trimer
as the coupling reaction proceeded. After coupling, SDS-PAGE
analysis showed the presence of monomer, dimer and trimer in
solution.
[0571] Purification of the Trimeric TAR1-5-19CYS
[0572] 40 .mu.L of 40% glacial acetic acid was added per mL of the
TMEA-TAR1-5-19cys reaction to reduce the pH to .about.4. The sample
was then applied to a 1 mL Resource S cation exchange column
(Amersham Pharmacia), which had been pre-equilibrated with 50 mM
sodium acetate pH 4.0. The dimer and trimer were partially
separated using a salt gradient of 340 to 450 mM Sodium chloride,
50 mM sodium acetate pH 4.0 over 30 column volumes. Fractions
containing trimer only were identified using SDS-PAGE and then
pooled and the pH increased to 8 by the addition of 1/5 volume of
1M Tris pH 8.0. To prevent precipitation of the trimer during
concentration steps (using 5K cut off Viva spin concentrators;
Vivascience), 10% glycerol was added to the sample.
[0573] In vitro Functional Binding Assay: TNF Receptor Assay and
Cell Assay
[0574] The affinity of the trimer for human TNF.alpha. was
determined using the TNF receptor and cell assay. IC50 in the
receptor assay was 0.3 nM; ND50 in the cell assay was in the range
of 3 to 10 nM (eg, 3 nM).
[0575] Other Possible TAR1-5-19CYS Trimer Formats
[0576] TAR1-5-19CYS may also be formatted into a trimer using the
following reagents:
[0577] PEG Trimers and Custom Synthetic Maleimide Trimers
[0578] Nektar (Shearwater) offer a range of multi arm PEGs, which
can be chemically modified at the terminal end of the PEG.
Therefore using a PEG trimer with a maleimide functional group at
the end of each arm would allow the trimerisation of the dAb in a
manner similar to that outlined above using TMEA. The PEG may also
have the advantage in increasing the solubility of the trimer thus
preventing the problem of aggregation. Thus, one could produce a
dAb trimer in which each dAb has a C-terminal cysteine that is
linked to a maleimide functional group, the maleimide functional
groups being linked to a PEG trimer.
[0579] Addition of a Polypeptide Linker or Hinge to the C-Terminus
of the dAb
[0580] A small linker, either (Gly.sub.4Ser).sub.n where n=1 to 10,
eg, 1, 2, 3, 4, 5, 6 or 7, an immunoglobulin (eg, IgG hinge region
or random peptide sequence (eg, selected from a library of random
peptide sequences) could be engineered between the dAb and the
terminal cysteine residue. When used to make multimers (eg, dimers
or trimers), this again would introduce a greater degree of
flexibility and distance between the individual monomers, which may
improve the binding characteristics to the target, eg a
multisubunit target such as human TNF.alpha..
EXAMPLE 9
Selection of a Collection of Single Domain Antibodies (dAbs)
Directed against Human Serum Albumin (HSA) and Mouse Serum Albumin
(MSA)
[0581] This example explains a method for making a single domain
antibody (dAb) directed against serum albumin. Selection of dAbs
against both mouse serum albumin (MSA) and human serum albumin
(HSA) is described. Three human phage display antibody libraries
were used in this experiment, each based on a single human
framework for V.sub.H (see FIG. 13: sequence of dummy V.sub.H based
on V3-23/DP47 and JH4b) or V.kappa. (see FIG. 15: sequence of dummy
V.kappa. based on o12/o2/DPK9 and Jk1) with side chain diversity
encoded by NNK codons incorporated in complementarity determining
regions (CDR1, CDR2 and CDR3).
[0582] Library 1 (V.sub.H):
[0583] Diversity at positions: H30, H31, H33, H35, H50, H52, H52a,
H53, H55, H56, H58, H95, H97, H98.
[0584] Library size: 6.2.times.10.sup.9
[0585] Library 2 (V.sub.H):
[0586] Diversity at positions: H30, H31, H33, H35, H50, H52, H52a,
H53, H55, H56, H58, H95, H97, H98, H99, H100, H100a, H100b.
[0587] Library size: 4.3.times.10.sup.9
[0588] Library 3 (V.kappa.):
[0589] Diversity at positions: L30, L31, L32, L34, L50, L53, L91,
L92, L93, L94, L96
[0590] Library size: 2.times.10.sup.9
[0591] The V.sub.H and V.kappa. libraries have been preselected for
binding to generic ligands protein A and protein L respectively so
that the majority of clones in the unselected libraries are
functional. The sizes of the libraries shown above correspond to
the sizes after preselection.
[0592] Two rounds of selection were performed on serum albumin
using each of the libraries separately. For each selection, antigen
was coated on immunotube (nunc) in 4 ml of PBS at a concentration
of 100 .mu.g/ml. In the first round of selection, each of the three
libraries was panned separately against HSA (Sigma) and MSA
(Sigma). In the second round of selection, phage from each of the
six first round selections was panned against (i) the same antigen
again (eg 1.sup.st round MSA, 2.sup.nd round MSA) and (ii) against
the reciprocal antigen (eg 1.sup.st round MSA, 2.sup.nd round HSA)
resulting in a total of twelve 2.sup.nd round selections. In each
case, after the second round of selection 48 clones were tested for
binding to HSA and MSA. Soluble dAb fragments were produced as
described for scFv fragments by Harrison et al, Methods Enzymol.
1996;267:83-109 and standard ELISA protocol was followed
(Hoogenboom et al. (1991) Nucleic Acids Res., 19: 4133) except that
2% tween PBS was used as a blocking buffer and bound dAbs were
detected with either protein L-HRP (Sigma) (for the V.kappa.S) and
protein A-HRP (Amersham Pharmacia Biotech) (for the V.sub.Hs).
[0593] dAbs that gave a signal above background indicating binding
to MSA, HSA or both were tested in ELISA insoluble form for binding
to plastic alone but all were specific for serum albumin. Clones
were then sequenced (see table below) revealing that 21 unique dAb
sequences had been identified. The minimum similarity (at the amino
acid level) between the V.kappa. dAb clones selected was 86.25%
((69/80).times.100; the result when all the diversified residues
are different, eg clones 24 and 34). The minimum similarity between
the V.sub.H dAb clones selected was 94% ((127/136).times.100).
[0594] Next, the serum albumin binding dAbs were tested for their
ability to capture biotinylated antigen from solution. ELISA
protocol (as above) was followed except that ELISA plate was coated
with 1 .mu.g/ml protein L (for the V.kappa. clones) and 1 .mu.g/ml
protein A (for the V.sub.H clones). Soluble dAb was captured from
solution as in the protocol and detection was with biotinylated MSA
or HSA and streptavidin HRP. The biotinylated MSA and HSA had been
prepared according to the manufacturer's instructions, with the aim
of achieving an average of 2 biotins per serum albumin molecule.
Twenty four clones were identified that captured biotinylated MSA
from solution in the ELISA. Two of these (clones 2 and 38 below)
also captured biotinylated HSA. Next, the dAbs were tested for
their ability to bind MSA coated on a CM5 biacore chip. Eight
clones were found that bound MSA on the biacore. TABLE-US-00011 dAb
(all Binds capture MSA Captures biotinylated H in biotinylated MSA)
or .kappa. CDR1 CDR2 CDR3 biacore? HSA? Vk library 3 .kappa. XXXLX
XASXLQS QQXXXXPXT template (SEQ ID NO: 133) (SEQ ID (SEQ ID (dummy)
NO: 134) NO: 135) 2, 4, 7, 41, .kappa. SSYLN RASPLQS QQTYSVPPT all
4 bind (SEQ ID NO: 136) (SEQ ID (SEQ ID NO: 137) NO: 138) 38, 54
.kappa. SSYLN RASPLQS QQTYRIPPT both bind (SEQ ID NO: 139) (SEQ ID
(SEQ ID NO: 140) NO: 141) 46, 47, 52, .kappa. FKSLK NASYLQS
QQVVYWPVT 56 (SEQ ID NO: 142) (SEQ ID (SEQ ID NO: 143) NO: 144) 13,
15 .kappa. YYHLK KASTLQS QQVRKVPRT (SEQ ID NO: 145) (SEQ ID (SEQ ID
NO: 146) NO: 147) 30, 35 .kappa. RRYLK QASVLQS QQGLYPPIT (SEQ ID
NO: 148) (SEQ ID (SEQ ID NO: 149) NO: 150) 19, .kappa. YNWLK
RASSLQS QQNVVIPRT (SEQ ID NO: 151) (SEQ ID (SEQ ID NO: 152) NO:
153) 22, .kappa. LWHLR HASLLQS QQSAVYPKT (SEQ ID NO: 154) (SEQ ID
(SEQ ID NO: 155) NO: 156) 23, .kappa. FRYLR HASHLQS QQRLLYPKT (SEQ
ID NO: 157) (SEQ ID (SEQ ID NO: 158) NO: 159) 24, .kappa. FYHLA
PASKLQS QQRARWPRT (SEQ ID NO: 160) (SEQ ID (SEQ ID NO: 161) NO:
162) 31, .kappa. IWHLN RASRLQS QQVARVPRT (SEQ ID NO: 163) (SEQ ID
(SEQ ID NO: 164) NO: 165) 33, .kappa. YRYLR KASSLQS QQYVGYPRT (SEQ
ID NO: 166) (SEQ ID (SEQ ID NO: 167) NO: 168) 34, .kappa. LKYLK
NASHLQS QQTTYYPIT (SEQ ID NO: 169) (SEQ ID (SEQ ID NO: 170) NO:
171) 53, .kappa. LRYLR KASWLQS QQVLYYPQT (SEQ ID NO: 172) (SEQ ID
(SEQ ID NO: 173) NO: 174) 11, .kappa. LRSLK AASRLQS QQVVYWPAT (SEQ
ID NO: 175) (SEQ ID (SEQ ID NO: 176) NO: 177) 12, .kappa. FRHLK
AASRLQS QQVALYPKT (SEQ ID NO: 178) (SEQ ID (SEQ ID NO: 179) NO:
180) 17, .kappa. RKYLR TASSLQS QQNLFWPRT (SEQ ID NO: 181) (SEQ ID
(SEQ ID NO: 182) NO: 183) 18, .kappa. RRYLN AASSLQS QQMLFYPKT (SEQ
ID NO: 184) (SEQ ID (SEQ ID NO: 185) NO: 186) 16, 21 .kappa. IKHLK
GASRLQS QQGARWPQT (SEQ ID NO: 187) (SEQ ID (SEQ ID NO: 188) NO:
189) 25, 26 .kappa. YYHLK KASTLQS QQVRKVPRT (SEQ ID NO: 190) (SEQ
ID (SEQ ID NO: 191) NO: 192) 27, .kappa. YKHLK NASHLQS QQVGRYPKT
(SEQ ID NO: 193) (SEQ ID (SEQ ID NO: 194) NO: 195) 55, .kappa.
FKSLK NASYLQS QQVVYWPVT (SEQ ID NO: 196) (SEQ ID (SEQ ID NO: 197)
NO: 198) V.sub.H library 1 H XXYXXX XIXXXGXXTXYADSVKG XXXX (XXXX)
FDY (and 2) (SEQ ID (SEQ ID NO: 200) (SEQ ID template NO: 199) NO:
201) (dummy) 8, 10 H WVYQMD SISAFGAKTLYADSVKG LSGKFDY (SEQ ID (SEQ
ID NO: 203) (SEQ ID NO: 202) NO: 204) 36, H WSYQMT
SISSFGSSTLYADSVKG GRDHNYSLFDY (SEQ ID (SEQ ID NO: 206) (SEQ ID NO:
205) NO: 207)
[0595] In all cases the frameworks were identical to the frameworks
in the corresponding dummy sequence, with diversity in the CDRs as
indicated in the table above.
[0596] Of the eight clones that bound MSA on the biacore, two
clones that are highly expressed in E. coli (clones MSA16 and
MSA26) were chosen for further study (see example 10). Full
nucleotide and amino acid sequences for MSA16 and 26 are given in
FIG. 16.
EXAMPLE 10
Determination of Affinity and Serum Half-Life in Mouse of MSA
Binding dAbs MSA16 and MSA26
[0597] dAbs MSA16 and MSA26 were expressed in the periplasm of E.
coli and purified using batch absorbtion to protein L-agarose
affinity resin (Affitech, Norway) followed by elution with glycine
at pH 2.2. The purified dAbs were then analysed by inhibition
biacore to determine K.sub.d. Briefly, purified MSA16 and MSA26
were tested to determine the concentration of dAb required to
achieve 200RUs of response on a biacore CM5 chip coated with a high
density of MSA. Once the required concentrations of dAb had been
determined, MSA antigen at a range of concentrations around the
expected K.sub.d was premixed with the dAb and incubated overnight.
Binding to the MSA coated biacore chip of dAb in each of the
premixes was then measured at a high flow-rate of 30 .mu.l/minute.
The resulting curves were used to create Klotz plots, which gave an
estimated K.sub.d of 200 nM for MSA16 and 70 nM for MSA 26 (FIG.
17A & B).
[0598] Next, clones MSA16 and MSA26 were cloned into an expression
vector with the HA tag (nucleic acid sequence:
TATCCTTATGATGTTCCTGATTATGCA (SEQ ID NO:216) and amino acid
sequence: YPYDVPDYA (SEQ ID NO:217)) and 2-10 mg quantities were
expressed in E. coli and purified from the supernatant with protein
L-agarose affinity resin (Affitech, Norway) and eluted with glycine
at pH2.2. Serum half life of the dAbs was determined in mouse.
MSA26 and MSA16 were dosed as single i.v. injections at approx 1.5
mg/kg into CD1 mice. Analysis of serum levels was by goat anti-HA
(Abcam, UK) capture and protein L-HRP (invitrogen) detection ELISA
which was blocked with 4% Marvel. Washing was with 0.05% tween PBS.
Standard curves of known concentrations of dAb were set up in the
presence of 1.times. mouse serum to ensure comparability with the
test samples. Modelling with a 2 compartment model showed MSA-26
had a t1/2.alpha. of 0.16 hr, a t1/2.beta. of 14.5 hr and an area
under the curve (AUC) of 465 hr.mg/ml (data not shown) and MSA-16
had a t1/2.alpha. of 0.98 hr, a t1/2.beta. of 36.5 hr and an AUC of
913 hr.mg/ml (FIG. 18). Both anti-MSA clones had considerably
lengthened half life compared with HEL4 (an anti-hen egg white
lysozyme dAb) which had a t1/2.alpha. of 0.06 hr, and a t1/2.beta.
of 0.34 hr.
EXAMPLE 11
Creation of V.sub.H-V.sub.H and V.kappa.-V.kappa. Dual Specific Fab
like Fragments
[0599] This example describes a method for making V.sub.H-V.sub.H
and V.kappa.-V.kappa. dual specifics as Fab like fragments. Before
constructing each of the Fab like fragments described, dAbs that
bind to targets of choice were first selected from dAb libraries
similar to those described in example 9. A V.sub.H dAb, HEL4, that
binds to hen egg lysozyme (Sigma) was isolated and a second V.sub.H
dAb (TAR2h-5) that binds to TNF.alpha. receptor (R and D systems)
was also isolated. The sequences of these are given in the sequence
listing. A V.kappa. dAb that binds TNF.alpha. (TAR1-5-19) was
isolated by selection and affinity maturation and the sequence is
also set forth in the sequence listing. A second V.kappa. dAb (MSA
26) described in example 9 whose sequence is in FIG. 17B was also
used in these experiments.
[0600] DNA from expression vectors containing the four dAbs
described above was digested with enzymes SalI and NotI to excise
the DNA coding for the dAb. A band of the expected size (300-400
bp) was purified by running the digest on an agarose gel and
excising the band, followed by gel purification using the Qiagen
gel purification kit (Qiagen, UK). The DNA coding for the dAbs was
then inserted into either the C.sub.H or C.kappa. vectors (FIGS. 8
and 9) as indicated in the table below. TABLE-US-00012 dAb V.sub.H
Target or dAb Inserted tag (C Antibiotic dAb antigen V.sub..kappa.
into vector terminal) resisitance HEL4 Hen egg V.sub.H C.sub.H myc
Chloram- lysozyme phenicol TAR2-5 TNF V.sub.H C.sub..kappa. flag
Ampicillin receptor TAR1-5- TNF .alpha. V.sub..kappa. C.sub.H myc
Chloram- 19 phenicol MSA 26 Mouse serum V.sub..kappa. C.sub..kappa.
flag Ampicillin albumin
[0601] The V.sub.H C.sub.H and V.sub.H C.kappa. constructs were
cotransformed into HB2151 cells. Separately, the V.kappa. C.sub.H
and V.kappa. C.kappa. constructs were cotransformed into HB2151
cells. Cultures of each of the cotransformed cell lines were grown
overnight (in 2.times. Ty containing 5% glucose, 10 .mu.g/ml
chloramphenicol and 100 .mu.g/ml ampicillin to maintain antibiotic
selection for both C.sub.H and C.kappa. plasmids). The overnight
cultures were used to inoculate fresh media (2.times. Ty, 10
.mu.g/ml chloramphenicol and 100 .mu.g/ml ampicillin) and grown to
OD 0.7-0.9 before induction by the addition of IPTG to express
their C.sub.H and C.kappa. constructs. Expressed Fab like fragment
was then purified from the periplasm by protein A purification (for
the contransformed V.sub.H C.sub.H and V.sub.H C.kappa.) and MSA
affinity resin purification (for the contransformed V.kappa.
C.sub.H and V.kappa. C.kappa.).
[0602] V.sub.H-V.sub.H Dual Specific
[0603] Expression of the V.sub.H C.sub.H and V.sub.H C.kappa. dual
specific was tested by running the protein on a gel. The gel was
blotted and a band the expected size for the Fab fragment could be
detected on the Western blot via both the myc tag and the flag tag,
indicating that both the V.sub.H C.sub.H and V.sub.H C.kappa. parts
of the Fab like fragment were present. Next, in order to determine
whether the two halves of the dual specific were present in the
same Fab-like fragment, an ELISA plate was coated overnight at
4.degree. C. with 100 .mu.l per well of hen egg lysozyme (HEL) at 3
mg/ml in sodium bicarbonate buffer. The plate was then blocked (as
described in example 1) with 2% tween PBS followed by incubation
with the V.sub.H C.sub.H/V.sub.H C.kappa. dual specific Fab like
fragment. Detection of binding of the dual specific to the HEL was
via the non cognate chain using 9e10 (a monoclonal antibody that
binds the myc tag, Roche) and anti mouse IgG-HRP (Amersham
Pharmacia Biotech). The signal for the V.sub.H C.sub.H/V.sub.H
C.kappa. dual specific Fab like fragment was 0.154 compared to a
background signal of 0.069 for the V.sub.H C.kappa. chain expressed
alone. This demonstrates that the Fab like fragment has binding
specificity for target antigen.
[0604] V.sub..kappa.-V.sub..kappa. Dual Specific
[0605] After purifying the contransformed V.kappa. C.sub.H and
V.kappa. C.kappa. dual specific Fab like fragment on an MSA
affinity resin, the resulting protein was used to probe an ELISA
plate coated with 1 .mu.g/ml TNF.alpha. and an ELISA plate coated
with 10 .mu.g/ml MSA. As predicted, there was signal above
background when detected with protein L-HRP on bot ELISA plates
(data not shown). This indicated that the fraction of protein able
to bind to MSA (and therefore purified on the MSA affinity column)
was also able to bind TNF.alpha. in a subsequent ELISA, confirming
the dual specificity of the antibody fragment. This fraction of
protein was then used for two subsequent experiments. Firstly, an
ELISA plate coated with 1 .mu.g/ml TNF.alpha. was probed with dual
specific V.kappa. C.sub.H and V.kappa. C.kappa. Fab like fragment
and also with a control TNF.alpha. binding dAb at a concentration
calculated to give a similar signal on the ELISA. Both the dual
specific and control dAb were used to probe the ELISA plate in the
presence and in the absence of 2 mg/ml MSA. The signal in the dual
specific well was reduced by more than 50% but the signal in the
dAb well was not reduced at all (see FIG. 19a). The same protein
was also put into the receptor assay with and without MSA and
competition by MSA was also shown (see FIG. 19c). This demonstrates
that binding of MSA to the dual specific is competitive with
binding to TNF.alpha..
EXAMPLE 12
Creation of a V.kappa.-V.kappa. Dual Specific cys Bonded Dual
Specific with Specificity for Mouse Serum Albumin and
TNF.alpha.
[0606] This example describes a method for making a dual specific
antibody fragment specific for both mouse serum albumin and
TNF.alpha. by chemical coupling via a disulphide bond. Both MSA16
(from example 1) and TAR1-5-19 dAbs were recloned into a pET based
vector with a C terminal cysteine and no tags. The two dAbs were
expressed at 4-10 mg levels and purified from the supernatant using
protein L-agarose affinity resin (Affitiech, Norway). The cysteine
tagged dAbs were then reduced with dithiothreitol. The TAR1-5-19
dAb was then coupled with dithiodipyridine to block reformation of
disulphide bonds resulting in the formation of PEP 1-5-19
homodimers. The two different dAbs were then mixed at pH 6.5 to
promote disulphide bond formation and the generation of TAR1-5-19,
MSA16 cys bonded heterodimers. This method for producing conjugates
of two unlike proteins was originally described by King et al.
(King T P, Li Y Kochoumian L Biochemistry. 1978 vol 17:1499-506
Preparation of protein conjugates via intermolecular disulfide bond
formation.) Heterodimers were separated from monomeric species by
cation exchange. Separation was confirmed by the presence of a band
of the expected size on a SDS gel. The resulting heterodimeric
species was tested in the TNF receptor assay and found to have an
IC50 for neutralising TNF of approximately 18 nM. Next, the
receptor assay was repeated with a constant concentration of
heterodimer (18 nM) and a dilution series of MSA and HSA. The
presence of HSA at a range of concentrations (up to 2 mg/ml) did
not cause a reduction in the ability of the dimer to inhibit
TNF.alpha.. However, the addition of MSA caused a dose dependant
reduction in the ability of the dimer to inhibit TNF.alpha. (FIG.
20). This demonstrates that MSA and TNF.alpha. compete for binding
to the cys bonded TAR1-5-19, MSA16 dimer.
[0607] Data Summary
[0608] A summary of data obtained in the experiments set forth in
the foregoing examples is set forth in Annex 4.
[0609] All publications mentioned in the present specification, and
references cited in said publications, are herein incorporated by
reference. Various modifications and variations of the described
methods and system of the invention will be apparent to those
skilled in the art without departing from the scope and spirit of
the invention. Although the invention has been described in
connection with specific preferred embodiments, it should be
understood that the invention as claimed should not be unduly
limited to such specific embodiments. Indeed, various modifications
of the described modes for carrying out the invention which are
obvious to those skilled in molecular biology or related fields are
intended to be within the scope of the following claims.
[0610] Annex 1; Polypeptides which Enhance Half-Life in vivo.
[0611] Alpha-1 Glycoprotein (Orosomucoid) (AAG)
[0612] Alpha-1 Antichyromotrypsin (ACT)
[0613] Alpha-1 Antitrypsin (AAT)
[0614] Alpha-1 Microglobulin (Protein HC) (AIM)
[0615] Alpha-2 Macroglobulin (A2M)
[0616] Antithrombin III (AT III)
[0617] Apolipoprotein A-1 (Apo A-1)
[0618] Apoliprotein B (Apo B)
[0619] Beta-2-microglobulin (B2M)
[0620] Ceruloplasmin (Cp)
[0621] Complement Component (C3)
[0622] Complement Component (C4)
[0623] C1 Esterase Inhibitor (C1 INH)
[0624] C-Reactive Protein (CRP)
[0625] Cystatin C (Cys C)
[0626] Ferritin (FER)
[0627] Fibrinogen (FIB)
[0628] Fibronectin (FN)
[0629] Haptoglobin (Hp)
[0630] Hemopexin (HPX)
[0631] Immunoglobulin A (IgA)
[0632] Immunoglobulin D (IgD)
[0633] Immunoglobulin E (IgE)
[0634] Immunoglobulin G (IgG)
[0635] Immunoglobulin M (IgM)
[0636] Immunoglobulin Light Chains (kapa/lambda)
[0637] Lipoprotein(a) [Lp(a)]
[0638] Mannose-bindign protein (MBP)
[0639] Myoglobin (Myo)
[0640] Plasminogen (PSM)
[0641] Prealbumin (Transthyretin) (PAL)
[0642] Retinol-binding protein (RBP)
[0643] Rheomatoid Factor (RF)
[0644] Serum Amyloid A (SAA)
[0645] Soluble Tranferrin Receptor (sTfR)
[0646] Transferrin (Tf) TABLE-US-00013 Annex 2 Pairing Therapeutic
relevant references. TNF TGF-b and TNF when injected into the ankle
joint of collagen ALPHA/TGF-.beta. induced arthritis model
significantly enhanced joint inflammation. In non-collagen
challenged mice there was no effect. TNF TNF and IL-1 synergize in
the pathology of uveitis. ALPHA/IL-1 TNF and IL-1 synergize in the
pathology of malaria (hypoglycaemia, NO). TNF and IL-1 synergize in
the induction of polymorphonuclear (PMN) cells migration in
inflammation. IL-1 and TNF synergize to induce PMN infiltration
into the peritoneum. IL-1 and TNF synergize to induce the secretion
of IL-1 by endothelial cells. Important in inflammation. IL-1 or
TNF alone induced some cellular infiltration into knee synovium.
IL-1 induced PMNs, TNF - monocytes. Together they induced a more
severe infiltration due to increased PMNs. Circulating myocardial
depressant substance (present in sepsis) is low levels of IL-1 and
TNFacting synergistically. TNF Most relating to synergisitic
activation of killer T-cells. ALPHA/IL-2 TNF Synergy of interleukin
3 and tumor necrosis factor alpha in ALPHA/IL-3 stimulating clonal
growth of acute myelogenous leukemia blasts is the result of
induction of secondary hematopoietic cytokines by tumor necrosis
factor alpha. Cancer Res. 1992 Apr 15; 52(8): 2197-201. TNF IL-4
and TNF synergize to induce VCAM expression on ALPHA/IL-4
endothelial cells. Implied to have a role in asthma. Same for
synovium - implicated in RA. TNF and IL-4 synergize to induce IL-6
expression in keratinocytes. Sustained elevated levels of VCAM-1 in
cultured fibroblast- like synoviocytes can be achieved by TNF-alpha
in combination with either IL-4 or IL-13 through increased mRNA
stability. Am J Pathol. 1999 Apr; 154(4): 1149-58 TNF Relationship
between the tumor necrosis factor system and the ALPHA/IL-5 serum
interleukin-4, interleukin-5, interleukin-8, eosinophil cationic
protein, and immunoglobulin E levels in the bronchial
hyperreactivity of adults and their children. Allergy Asthma Proc.
2003 Mar-Apr; 24(2): 111-8. TNF TNF and IL-6 are potent growth
factors for OH-2, a novel ALPHA/IL-6 human myeloma cell line. Eur J
Haematol. 1994 Jul; 53(1): 31-7. TNF TNF and IL-8 synergized with
PMNs to activate platelets. ALPHA/IL-8 Implicated in Acute
Respiratory Distress Syndrome. See IL-5/TNF (asthma). Synergism
between interleukin-8 and tumor necrosis factor-alpha for
neutrophil-mediated platelet activation. Eur Cytokine Netw. 1994
Sep-Oct; 5(5): 455-60. (adult respiratory distress syndrome (ARDS))
TNF ALPHA/IL-9 TNF IL-10 induces and synergizes with TNF in the
induction of ALPHA/IL-10 HIV expression in chronically infected
T-cells. TNF Cytokines synergistically induce osteoclast
differentiation: ALPHA/IL-11 support by immortalized or normal
calvarial cells. Am J Physiol Cell Physiol. 2002 Sep; 283(3):
C679-87. (Bone loss) TNF ALPHA/IL-12 TNF Sustained elevated levels
of VCAM-1 in cultured fibroblast- ALPHA/IL-13 like synoviocytes can
be achieved by TNF-alpha in combination with either IL-4 or IL-13
through increased mRNA stability. Am J Pathol. 1999 Apr; 154(4):
1149-58. Interleukin-13 and tumour necrosis factor-alpha
synergistically induce eotaxin production in human nasal
fibroblasts. Clin Exp Allergy. 2000 Mar; 30(3): 348-55.
Interleukin-13 and tumour necrosis factor-alpha synergistically
induce eotaxin production in human nasal fibroblasts. Clin Exp
Allergy. 2000 Mar; 30(3): 348-55 (allergic inflammation)
Implications of serum TNF-beta and IL-13 in the treatment response
of childhood nephrotic syndrome. Cytokine. 2003 Feb 7; 21(3):
155-9. TNF Effects of inhaled tumour necrosis factor alpha in
subjects with ALPHA/IL-14 mild asthma. Thorax. 2002 Sep; 57(9):
774-8. TNF Effects of inhaled tumour necrosis factor alpha in
subjects with ALPHA/IL-15 mild asthma. Thorax. 2002 Sep; 57(9):
774-8. TNF Tumor necrosis factor-alpha-induced synthesis of
interleukin- ALPHA/IL-16 16 in airway epithelial cells: priming for
serotonin stimulation. Am J Respir Cell Mol Biol. 2003 Mar; 28(3):
354-62. (airway inflammation) Correlation of circulating
interleukin 16 with proinflammatory cytokines in patients with
rheumatoid arthritis. Rheumatology (Oxford). 2001 Apr; 40(4):
474-5. No abstract available. Interleukin 16 is up-regulated in
Crohn's disease and participates in TNBS colitis in mice.
Gastroenterology. 2000 Oct; 119(4): 972-82. TNF Inhibition of
interleukin-17 prevents the development of ALPHA/IL-17 arthritis in
vaccinated mice challenged with Borrelia burgdorferi. Infect Immun.
2003 Jun; 71(6): 3437-42. Interleukin 17 synergises with tumour
necrosis factor alpha to induce cartilage destruction in vitro. Ann
Rheum Dis. 2002 Oct; 61(10): 870-6. A role of GM-CSF in the
accumulation of neutrophils in the airways caused by IL-17 and
TNF-alpha. Eur Respir J. 2003 Mar; 21(3): 387-93. (Airway
inflammation) Abstract Interleukin-1, tumor necrosis factor alpha,
and interleukin-17 synergistically up-regulate nitric oxide and
prostaglandin E2 production in explants of human osteoarthritic
knee menisci. Arthritis Rheum. 2001 Sep; 44(9): 2078-83. TNF
Association of interleukin-18 expression with enhanced levels
ALPHA/IL-18 of both interleukin-1beta and tumor necrosis factor
alpha in knee synovial tissue of patients with rheumatoid
arthritis. Arthritis Rheum. 2003 Feb; 48(2): 339-47. Abstract
Elevated levels of interleukin-18 and tumor necrosis factor-alpha
in serum of patients with type 2 diabetes mellitus: relationship
with diabetic nephropathy. Metabolism. 2003 May; 52(5): 605-8. TNF
Abstract IL-19 induces production of IL-6 and TNF-alpha and
ALPHA/IL-19 results in cell apoptosis through TNF-alpha. J Immunol.
2002 Oct 15; 169(8): 4288-97. TNF Abstract Cytokines: IL-20 - a new
effector in skin ALPHA/IL-20 inflammation. Curr Biol. 2001 Jul 10;
11(13): R531-4 TNF Inflammation and coagulation: implications for
the septic ALPHA/Complement patient. Clin Infect Dis. 2003 May 15;
36(10): 1259-65. Epub 2003 May 08. Review. TNF MHC induction in the
brain. ALPHA/IFN-.gamma. Synergize in anti-viral
response/IFN-.beta. induction. Neutrophil activation/respiratory
burst. Endothelial cell activation Toxicities noted when patients
treated with TNF/IFN-.gamma. as anti- viral therapy Fractalkine
expression by human astrocytes. Many papers on inflammatory
responses - i.e. LPS, also macrophage activation. Anti-TNF and
anti-IFN-.gamma. synergize to protect mice from lethal endotoxemia.
TGF-.beta./IL-1 Prostaglndin synthesis by osteoblasts IL-6
production by intestinal epithelial cells (inflammation model)
Stimulates IL-11 and IL-6 in lung fibroblasts (inflammation model)
IL-6 and IL-8 production in the retina TGF-.beta./IL-6
Chondrocarcoma proliferation IL-1/IL-2 B-cell activation LAK cell
activation T-cell activation IL-1 synergy with IL-2 in the
generation of lymphokine activated killer cells is mediated by
TNF-alpha and beta (lymphotoxin). Cytokine. 1992 Nov; 4(6): 479-87.
IL-1/IL-3 IL-1/IL-4 B-cell activation IL-4 induces IL-1 expression
in endothelial cell activation. IL-1/IL-5 IL-1/IL-6 B cell
activation T cell activation (can replace accessory cells) IL-1
induces IL-6 expression C3 and serum amyloid expression (acute
phase response) HIV expression Cartilage collagen breakdown.
IL-1/IL-7 IL-7 is requisite for IL-1-induced thymocyte
proliferation. Involvement of IL-7 in the synergistic effects of
granulocyte- macrophage colony-stimulating factor or tumor necrosis
factor with IL-1. J Immunol. 1992 Jan 1; 148(1): 99-105. IL-1/IL-8
IL-1/IL-10 IL-1/IL-11 Cytokines synergistically induce osteoclast
differentiation: support by immortalized or normal calvarial cells.
Am J Physiol Cell Physiol. 2002 Sep; 283(3): C679-87. (Bone loss)
IL-1/IL-16 Correlation of circulating interleukin 16 with
proinflammatory cytokines in patients with rheumatoid arthritis.
Rheumatology (Oxford). 2001 Apr; 40(4): 474-5. No abstract
available. IL-1/IL-17 Inhibition of interleukin-17 prevents the
development of arthritis in vaccinated mice challenged with
Borrelia burgdorferi. Infect Immun. 2003 Jun; 71(6): 3437-42.
Contribution of interleukin 17 to human cartilage degradation and
synovial inflammation in osteoarthritis. Osteoarthritis Cartilage.
2002 Oct; 10(10): 799-807. Abstract Interleukin-1, tumor necrosis
factor alpha, and interleukin-17 synergistically up-regulate nitric
oxide and prostaglandin E2 production in explants of human
osteoarthritic knee menisci. Arthritis Rheum. 2001 Sep; 44(9):
2078-83. IL-1/IL-18 Association of interleukin-18 expression with
enhanced levels of both interleukin-1beta and tumor necrosis factor
alpha in knee synovial tissue of patients with rheumatoid
arthritis. Arthritis Rheum. 2003 Feb; 48(2): 339-47. IL-1/IFN-g
IL-2/IL-3 T-cell proliferation B cell proliferation IL-2/IL-4
B-cell proliferation T-cell proliferation (selectively inducing
activation of CD8 and NK lymphocytes)IL-2R beta agonist P1-30 acts
in synergy with IL-2, IL-4, IL-9, and IL-15: biological and
molecular effects. J Immunol. 2000 Oct 15; 165(8): 4312-8.
IL-2/IL-5 B-cell proliferation/Ig secretion IL-5 induces IL-2
receptors on B-cells IL-2/IL-6 Development of cytotoxic T-cells
IL-2/IL-7 IL-2/IL-9 See IL-2/IL-4 (NK-cells) IL-2/IL-10 B-cell
activation IL-2/IL-12 IL-12 synergizes with IL-2 to induce
lymphokine-activated cytotoxicity and perforin and granzyme gene
expression in fresh human NK cells. Cell Immunol. 1995 Oct 1;
165(1): 33-43. (T-cell activation) IL-2/IL-15 See IL-2/IL-4 (NK
cells) (T cell activation and proliferation) IL-15 and IL-2: a
matter of life and death for T cells in vivo. Nat Med. 2001 Jan;
7(1): 114-8. IL-2/IL-16 Synergistic activation of CD4+ T cells by
IL-16 and IL-2. J Immunol. 1998 Mar 1; 160(5): 2115-20. IL-2/IL-17
Evidence for the early involvement of interleukin 17 in human and
experimental renal allograft rejection. J Pathol. 2002 Jul; 197(3):
322-32. IL-2/IL-18 Interleukin 18 (IL-18) in synergy with IL-2
induces lethal lung injury in mice: a potential role for cytokines,
chemokines, and natural killer cells in the pathogenesis of
interstitial pneumonia. Blood. 2002 Feb 15; 99(4): 1289-98.
IL-2/TGF-.beta. Control of CD4 effector fate: transforming growth
factor beta 1 and interleukin 2 synergize to prevent apoptosis and
promote effector expansion. J Exp Med. 1995 Sep 1; 182(3): 699-709.
IL-2/IFN-.gamma. Ig secretion by B-cells IL-2 induces IFN-.gamma.
expression by T-cells IL-2/IFN-.alpha./.beta. None IL-3/IL-4
Synergize in mast cell growth Synergistic effects of IL-4 and
either GM-CSF or IL-3 on the induction of CD23 expression by human
monocytes: regulatory effects of IFN-alpha and IFN-gamma. Cytokine.
1994 Jul; 6(4): 407-13. IL-3/IL-5 IL-3/IL-6 IL-3/IFN-.gamma. IL-4
and IFN-gamma synergistically increase total polymeric IgA receptor
levels in human intestinal epithelial cells. Role of protein
tyrosine kinases. J Immunol. 1996 Jun 15; 156(12): 4807-14.
IL-3/GM-CSF Differential regulation of human eosinophil IL-3, IL-5,
and GM-CSF receptor alpha-chain expression by cytokines: IL-3,
IL-5, and GM-CSF down-regulate IL-5 receptor alpha expression with
loss of IL-5 responsiveness, but up-regulate IL-3 receptor alpha
expression. J Immunol. 2003 Jun 1; 170(11): 5359-66. (allergic
inflammation) IL-4/IL-2 IL-4 synergistically enhances both IL-2-
and IL-12-induced IFN-{gamma} expression in murine NK cells. Blood.
2003 Mar 13 [Epub ahead of print] IL-4/IL-5 Enhanced mast cell
histamine etc. secretion in response to IgE A Th2-like cytokine
response is involved in bullous pemphigoid. the role of IL-4 and
IL-5 in the pathogenesis of the disease. Int J Immunopathol
Pharmacol. 1999 May-Aug; 12(2): 55-61. IL-4/IL-6 IL-4/IL-10
IL-4/IL-11 Synergistic interactions between interleukin-11 and
interleukin-4 in support of proliferation of primitive
hematopoietic progenitors of mice. Blood. 1991 Sep 15; 78(6):
1448-51. IL-4/IL-12 Synergistic effects of IL-4 and IL-18 on
IL-12-dependent IFN- gamma production by dendritic cells. J
Immunol. 2000 Jan 1; 164(1): 64-71. (increase Th1/Th2
differentiation) IL-4 synergistically enhances both IL-2- and
IL-12-induced IFN-{gamma} expression in murine NK cells. Blood.
2003 Mar 13 [Epub ahead of print] IL-4/IL-13 Abstract Interleukin-4
and interleukin-13 signaling connections maps. Science. 2003 Jun 6;
300(5625): 1527-8. (allergy, asthma) Inhibition of the IL-4/IL-13
receptor system prevents allergic sensitization without affecting
established allergy in a mouse model for allergic asthma. J Allergy
Clin Immunol. 2003 Jun; 111(6): 1361-1369. IL-4/IL-16 (asthma)
Interleukin (IL)-4/IL-9 and exogenous IL-16 induce IL-16 production
by BEAS-2B cells, a bronchial epithelial cell line. Cell Immunol.
2001 Feb 1; 207(2): 75-80 IL-4/IL-17 Interleukin (IL)-4 and IL-17
synergistically stimulate IL-6 secretion in human colonic
myofibroblasts. Int J Mol Med. 2002 Nov; 10(5): 631-4. (Gut
inflammation) IL-4/IL-24 IL-24 is expressed by rat and human
macrophages. Immunobiology. 2002 Jul; 205(3): 321-34. IL-4/IL-25
Abstract New IL-17 family members promote Th1 or Th2 responses in
the lung: in vivo function of the novel cytokine IL-25. J Immunol.
2002 Jul 1; 169(1): 443-53. (allergic inflammation) Abstract Mast
cells produce interleukin-25 upon Fcepsilon RI- mediated
activation. Blood. 2003 May 1; 101(9): 3594-6. Epub 2003 Jan 02.
(allergic inflammation) IL-4/IFN-.gamma. Abstract Interleukin 4
induces interleukin 6 production by endothelial cells: synergy with
interferon-gamma. Eur J Immunol. 1991 Jan; 21(1): 97-101. IL-4/SCF
Regulation of human intestinal mast cells by stem cell factor and
IL-4. Immunol Rev. 2001 Feb; 179: 57-60. Review. IL-5/IL-3
Differential regulation of human eosinophil IL-3, IL-5, and GM-CSF
receptor alpha-chain expression by cytokines: IL-3, IL-5, and
GM-CSF down-regulate IL-5 receptor alpha expression with loss of
IL-5 responsiveness, but up-regulate IL-3 receptor alpha
expression. J Immunol. 2003 Jun 1; 170(11): 5359-66. (Allergic
inflammation see abstract) IL-5/IL-6 IL-5/IL-13 Inhibition of
allergic airways inflammation and airway hyperresponsiveness in
mice by dexamethasone: role of eosinophils, IL-5, eotaxin, and
IL-13. J Allergy Clin Immunol. 2003 May; 111(5): 1049-61.
IL-5/IL-17 Interleukin-17 orchestrates the granulocyte influx into
airways after allergen inhalation in a mouse model of allergic
asthma. Am J Respir Cell Mol Biol. 2003 Jan; 28(1): 42-50.
IL-5/IL-25 Abstract New IL-17 family members promote Th1 or Th2
responses in the lung: in vivo function of the novel cytokine
IL-25. J Immunol. 2002 Jul 1; 169(1): 443-53. (allergic
inflammation) Abstract Mast cells produce interleukin-25 upon
Fcepsilon RI- mediated activation. Blood. 2003 May 1; 101(9):
3594-6. Epub 2003 Jan 02. (allergic inflammation) IL-5/IFN-.gamma.
IL-5/GM-CSF Differential regulation of human eosinophil IL-3, IL-5,
and GM-CSF receptor alpha-chain expression by cytokines: IL-3,
IL-5, and GM-CSF down-regulate IL-5 receptor alpha expression with
loss of IL-5 responsiveness, but up-regulate IL-3 receptor alpha
expression. J Immunol. 2003 Jun 1; 170(11): 5359-66. (Allergic
inflammation) IL-6/IL-10 IL-6/IL-11 IL-6/IL-16 Interleukin-16
stimulates the expression and production of pro- inflammatory
cytokines by human monocytes. Immunology. 2000 May; 100(1): 63-9.
IL-6/IL-17 Stimulation of airway mucin gene expression by
interleukin (IL)-17 through IL-6 paracrine/autocrine loop. J Biol
Chem. 2003 May 9; 278(19): 17036-43. Epub 2003 Mar 06. (airway
inflammation, asthma) IL-6/IL-19 Abstract IL-19 induces production
of IL-6 and TNF-alpha and results in cell apoptosis through
TNF-alpha. J Immunol. 2002 Oct 15; 169(8): 4288-97. IL-6/IFN-g
IL-7/IL-2 Interleukin 7 worsens graft-versus-host disease. Blood.
2002 Oct 1; 100(7): 2642-9. IL-7/IL-12 Synergistic effects of IL-7
and IL-12 on human T cell activation. J Immunol. 1995 May 15;
154(10): 5093-102. IL-7/IL-15 Interleukin-7 and interleukin-15
regulate the expression of the bcl-2 and c-myb genes in cutaneous
T-cell lymphoma cells. Blood. 2001 Nov 1; 98(9): 2778-83. (growth
factor) IL-8/IL-11 Abnormal production of interleukin (IL)-11 and
IL-8 in polycythaemia vera. Cytokine. 2002 Nov 21; 20(4): 178-83.
IL-8/IL-17 The Role of IL-17 in Joint Destruction. Drug News
Perspect. 2002 Jan; 15(1): 17-23. (arthritis) Abstract
Interleukin-17 stimulates the expression of interleukin-8,
growth-related oncogene-alpha, and granulocyte-colony-stimulating
factor by human airway epithelial cells. Am J Respir Cell Mol Biol.
2002 Jun; 26(6): 748-53. (airway inflammation) IL-8/GSF
Interleukin-8: an autocrine/paracrine growth factor for human
hematopoietic progenitors acting in synergy with colony stimulating
factor-1 to promote monocyte-macrophage growth and differentiation.
Exp Hematol. 1999 Jan; 27(1): 28-36. IL-8/VGEF Intracavitary VEGF,
bFGF, IL-8, IL-12 levels in primary and recurrent malignant glioma.
J Neurooncol. 2003 May; 62(3): 297-303. IL-9/IL-4
Anti-interleukin-9 antibody treatment inhibits airway inflammation
and hyperreactivity in mouse asthma model. Am J Respir Crit Care
Med. 2002 Aug 1; 166(3): 409-16. IL-9/IL-5 Pulmonary overexpression
of IL-9 induces Th2 cytokine expression, leading to immune
pathology. J Clin Invest. 2002 Jan; 109(1): 29-39. Th2 cytokines
and asthma. Interleukin-9 as a therapeutic target for asthma.
Respir Res. 2001; 2(2): 80-4. Epub 2001 Feb 15. Review. Abstract
Interleukin-9 enhances interleukin-5 receptor expression,
differentiation, and survival of human eosinophils. Blood. 2000 Sep
15; 96(6): 2163-71 (asthma) IL-9/IL-13 Anti-interleukin-9 antibody
treatment inhibits airway inflammation and hyperreactivity in mouse
asthma model. Am J Respir Crit Care Med. 2002 Aug 1; 166(3):
409-16. Direct effects of interleukin-13 on epithelial cells cause
airway hyperreactivity and mucus overproduction in asthma. Nat Med.
2002 Aug; 8(8): 885-9. IL-9/IL-16 See IL-4/IL-16 IL-10/IL-2 The
interplay of interleukin-10 (IL-10) and interleukin-2 (IL- 2) in
humoral immune responses: IL-10 synergizes with IL-2 to enhance
responses of human B lymphocytes in a mechanism which is different
from upregulation of CD25 expression. Cell Immunol. 1994 Sep;
157(2): 478-88. IL-10/IL-12 IL-10/TGF-.beta. IL-10 and TGF-beta
cooperate in the regulatory T cell response to mucosal allergens in
normal immunity and specific immunotherapy. Eur J Immunol. 2003
May; 33(5): 1205-14. IL-10/IFN-.gamma. IL-11/IL-6 Interleukin-6 and
interleukin-11 support human osteoclast formation by a
RANKL-independent mechanism. Bone. 2003 Jan; 32(1): 1-7. (bone
resorption in inflammation) IL-11/IL-17 Polarized in vivo
expression of IL-11 and IL-17 between acute and chronic skin
lesions. J Allergy Clin Immunol. 2003 Apr; 111(4): 875-81.
(allergic dermatitis) IL-17 promotes bone erosion in murine
collagen-induced arthritis through loss of the receptor activator
of NF-kappa B ligand/osteoprotegerin balance. J Immunol. 2003 Mar
1; 170(5): 2655-62. IL-11/TGF-.beta. Polarized in vivo expression
of IL-11 and IL-17 between acute and chronic skin lesions. J
Allergy Clin Immunol. 2003 Apr; 111(4): 875-81. (allergic
dermatitis) IL-12/IL-13 Relationship of Interleukin-12 and
Interleukin-13 imbalance with class-specific rheumatoid factors and
anticardiolipin antibodies in systemic lupus erythematosus. Clin
Rheumatol. 2003 May; 22(2): 107-11. IL-12/IL-17 Upregulation of
interleukin-12 and -17 in active inflammatory bowel disease. Scand
J Gastroenterol. 2003 Feb; 38(2): 180-5. IL-12/IL-18 Synergistic
proliferation and activation of natural killer cells by interleukin
12 and interleukin 18. Cytokine. 1999 Nov; 11(11): 822-30.
Inflammatory Liver Steatosis Caused by IL-12 and IL-18. J
Interferon Cytokine Res. 2003 Mar; 23(3): 155-62. IL-12/IL-23
nterleukin-23 rather than interleukin-12 is the critical cytokine
for autoimmune inflammation of the brain. Nature. 2003 Feb 13;
421(6924): 744-8. Abstract A unique role for IL-23 in promoting
cellular immunity. J Leukoc Biol. 2003 Jan; 73(1): 49-56. Review.
IL-12/IL-27 Abstract IL-27, a heterodimeric cytokine composed of
EBI3 and p28 protein, induces proliferation of naive CD4(+) T
cells. Immunity. 2002 Jun; 16(6): 779-90. IL-12/IFN-.gamma. IL-12
induces IFN-.gamma. expression by B and T-cells as part of immune
stimulation. IL-13/IL-5 See IL-5/IL-13 IL-13/IL-25 Abstract New
IL-17 family members promote Th1 or Th2 responses in the lung: in
vivo function of the novel cytokine IL-25. J Immunol. 2002 Jul 1;
169(1): 443-53. (allergic inflammation) Abstract Mast cells produce
interleukin-25 upon Fcepsilon RI- mediated activation. Blood. 2003
May 1; 101(9): 3594-6. Epub 2003 Jan 02. (allergic inflammation)
IL-15/IL-13 Differential expression of interleukins (IL)-13 and
IL-15 in ectopic and eutopic endometrium of women with
endometriosis and normal fertile women. Am J Reprod Immunol. 2003
Feb; 49(2): 75-83. IL-15/IL-16 IL-15 and IL-16 overexpression in
cutaneous T-cell lymphomas: stage-dependent increase in mycosis
fungoides progression. Exp Dermatol. 2000 Aug; 9(4): 248-51.
IL-15/IL-17 Abstract IL-17, produced by lymphocytes and
neutrophils, is necessary for lipopolysaccharide-induced airway
neutrophilia: IL-15 as a possible trigger. J Immunol. 2003 Feb 15;
170(4): 2106-12. (airway inflammation) IL-15/IL-21 IL-21 in Synergy
with IL-15 or IL-18 Enhances IFN-gamma Production in Human NK and T
Cells. J Immunol. 2003 Jun 1; 170(11): 5464-9. IL-17/IL-23
Interleukin-23 promotes a distinct CD4 T cell activation state
characterized by the production of interleukin-17. J Biol Chem.
2003 Jan 17; 278(3): 1910-4. Epub 2002 Nov 03 IL-17/TGF-.beta.
Polarized in vivo expression of IL-11 and IL-17 between acute and
chronic skin lesions. J Allergy Clin Immunol. 2003 Apr; 111(4):
875-81. (allergic dermatitis) IL-18/IL-12 Synergistic proliferation
and activation of natural killer cells by interleukin 12 and
interleukin 18. Cytokine. 1999 Nov; 11(11): 822-30. Abstract
Inhibition of in vitro immunoglobulin production by IL-12 in murine
chronic graft-vs.-host disease: synergism with IL-18. Eur J
Immunol. 1998 Jun; 28(6): 2017-24. IL-18/IL-21 IL-21 in Synergy
with IL-15 or IL-18 Enhances IFN-gamma Production in Human NK and T
Cells. J Immunol. 2003 Jun 1; 170(11): 5464-9. IL-18/TGF-.beta.
Interleukin 18 and transforming growth factor betal in the serum of
patients with Graves' ophthalmopathy treated with corticosteroids.
Int Immunopharmacol. 2003 Apr; 3(4): 549-52. IL-18/IFN-.gamma.
Anti-TNF Synergistic therapeutic effect in DBA/1 arthritic mice.
ALPHA/anti- CD4
[0647] TABLE-US-00014 Annex 3: Oncology combinations Target Disease
Pair with CD89* Use as cytotoxic cell all recruiter CD19 B cell
lymphomas HLA-DR CD5 HLA-DR B cell lymphomas CD89 CD19 CD5 CD38
Multiple myeloma CD138 CD56 HLA-DR CD138 Multiple myeloma CD38 CD56
HLA-DR CD138 Lung cancer CD56 CEA CD33 Acute myelod lymphoma CD34
HLA-DR CD56 Lung cancer CD138 CEA CEA Pan carcinoma MET receptor
VEGF Pan carcinoma MET receptor VEGF Pan carcinoma MET receptor
receptor IL-13 Asthma/pulmonary IL-4 inflammation IL-5 Eotaxin(s)
MDC TARC TNF.alpha. IL-9 EGFR CD40L IL-25 MCP-1 TGF.beta. IL-4
Asthma IL-13 IL-5 Eotaxin(s) MDC TARC TNF.alpha. IL-9 EGFR CD40L
IL-25 MCP-1 TGF.beta. Eotaxin Asthma IL-5 Eotaxin-2 Eotaxin-3 EGFR
cancer HER2/neu HER3 HER4 HER2 cancer HER3 HER4 TNFR1 RA/Crohn's
disease IL-1R IL-6R IL-18R TNF.alpha. RA/Crohn's disease
IL-1.alpha./.beta. IL-6 IL-18 ICAM-1 IL-15 IL-17 IL-1R RA/Crohn's
disease IL-6R IL-18R IL-18R RA/Crohn's disease IL-6R
[0648] TABLE-US-00015 Annex 4 Data Summary Equilibrium dissocation
constant ND50 for cell based TARGET dAb (Kd = Koff/Kon) Koff IC50
for ligand assay neutralisn assay TAR1 TAR1 300 nM to 5 pM 5
.times. 10.sup.-1 to 1 .times. 10.sup.-7 500 nM to 100 pM 500 nM to
50 pM monomers (ie, 3 .times. 10.sup.-7 to 5 .times. 10.sup.-12),
preferably 50 nM to 20 pM TAR1 As TAR1 monomer As TAR1 monomer As
TAR1 monomer As TAR1 monomer dimers TAR1 As TAR1 monomer As TAR1
monomer As TAR1 monomer As TAR1 monomer trimers TAR1-5 TAR1-27
TAR1-5-19 30 nM monomer TAR1-5-19 With (Gly.sub.4Ser).sub.3 linker
= 20 nm =30 nM homodimer With (Gly.sub.4Ser).sub.5 linker = 2 nm =3
nM With (Gly.sub.4Ser).sub.7 linker = 10 nm =15 nM In Fab format =
1 nM TAR1-5-19 With (Gly.sub.4Ser).sub.n linker =12 nM heterodimers
TAR1-5-19 d2 = 2 nM TAR1-5-19 d3 = 8 nM TAR1-5-19 d4 = 2-5 nM
TAR1-5-19 d5 = 8 nM =10 nM In Fab format =12 nM TAR1-5-19CH d1CK =
6 nM TAR1-5-19CK d1CH = 6 nM TAR1-5-19CH d2CK = 8 nM TAR1-5-19CH
d3CK = 3 nM TAR1-5 With (Gly.sub.4Ser).sub.n linker heterodimers
TAR1-5d1 = 30 nM TAR1-5d2 = 50 nM TAR1-5d3 = 300 nM TAR1-5d4 = 3 nM
TAR1-5d5 = 200 nM TAR1-5d6 = 100 nM In Fab format =60 nM TAR1-5CH
d2CK = 30 nM TAR1-5CK d3CH = 100 nM TAR1-5-19 0.3 nM 3-10 nM (eg, 3
nM) homotrimer TAR2 TAR2 As TAR1 monomer As TAR1 monomer 500 nM to
100 pM 500 nM to 50 pM monomers TAR2-10 TAR2-5 Serum Anti-SA 1 nM
to 500 .mu.M, 1 nM to 500 .mu.M, Albumin monomers preferably 100 nM
to preferably 100 nM to 10 .mu.M 10 .mu.M In Dual Specific In Dual
Specific format, format, target affinity target affinity is 1 to is
1 to 100,000 .times. affinity 100,000 .times. affinity of SA of SA
dAb dAb affinity, eg 100 pM affinity, eg 100 pM (target) and 10
.mu.M SA (target) and 10 .mu.M SA affinity. affinity. MSA-16 200 nM
MSA-26 70 nM
[0649]
Sequence CWU 1
1
218 1 720 DNA Homo sapiens 1 gaggtgcagc tgttggagtc tgggggaggc
ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt
cacctttagc agctatgcca tgagctgggt ccgccaggct 120 ccagggaagg
ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180
gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat
240 ctgcaaatga acagcctgag agccgaggac acggccgtat attactgtgc
gaaaagttat 300 ggtgcttttg actactgggg ccagggaacc ctggtcaccg
tctcgagcgg tggaggcggt 360 tcaggcggag gtggcagcgg cggtggcggg
tcgacggaca tccagatgac ccagtctcca 420 tcctccctgt ctgcatctgt
aggagacaga gtcaccatca cttgccgggc aagtcagagc 480 attagcagct
atttaaattg gtatcagcag aaaccaggga aagcccctaa gctcctgatc 540
tatgctgcat ccagtttgca aagtggggtc ccatcaaggt tcagtggcag tggatctggg
600 acagatttca ctctcaccat cagcagtctg caacctgaag attttgcaac
ttactactgt 660 caacagagtt acagtacccc taatacgttc ggccaaggga
ccaaggtgga aatcaaacgg 720 2 240 PRT Homo sapiens 2 Glu Val Gln Leu
Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser
Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ser Tyr Gly Ala Phe Asp Tyr
Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125 Gly Gly Ser Thr Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser 130 135 140 Ala Ser Val
Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser 145 150 155 160
Ile Ser Ser Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 165
170 175 Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro
Ser 180 185 190 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser 195 200 205 Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr
Cys Gln Gln Ser Tyr 210 215 220 Ser Thr Pro Asn Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys Arg 225 230 235 240 3 359 DNA Artificial
Sequence phagemid pIT1/pIT2 3 caggaaacag ctatgaccat gattacgcca
agcttgcatg caaattctat ttcaaggaga 60 cagtcataat gaaataccta
ttgcctacgg cagccgctgg attgttatta ctcgcggccc 120 agccggccat
ggccgaggtg tttgactact ggggccaggg aaccctggtc accgtctcga 180
gcggtggagg cggttcaggc ggaggtggca gcggcggtgg cgggtcgacg gacatccaga
240 tgacccaggc ggccgcagaa caaaaactcc atcatcatca ccatcacggg
gccgcaatct 300 cagaagagga tctgaatggg gccgcataga ctgttgaaag
ttgtttagca aaacctcat 359 4 96 PRT Artificial Sequence phagemid
pIT1/pIT2 4 Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu
Leu Ala 1 5 10 15 Ala Gln Pro Ala Met Ala Glu Val Phe Asp Tyr Trp
Gly Gln Gly Thr 20 25 30 Leu Val Thr Val Ser Ser Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser 35 40 45 Gly Gly Gly Gly Ser Thr Asp Ile
Gln Met Thr Gln Ala Ala Ala Glu 50 55 60 Gln Lys Leu His His His
His His His Gly Ala Ala Ile Ser Glu Glu 65 70 75 80 Asp Leu Asn Gly
Ala Ala Thr Val Glu Ser Cys Leu Ala Lys Pro His 85 90 95 5 116 PRT
Homo sapiens 5 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly
Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala
Lys Ser Tyr Gly Ala Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105
110 Thr Val Ser Ser 115 6 116 PRT Homo sapiens 6 Glu Val Gln Leu
Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Ser His Ile Ser Pro Tyr Gly Ala Asn Thr Arg Tyr Ala Asp Ser
Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Gly Leu Arg Ala Phe Asp Tyr
Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 7 116
PRT Homo sapiens 7 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val
Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Asp Ile Gly Ala Thr
Gly Ser Lys Thr Gly Tyr Ala Asp Pro Val 50 55 60 Lys Gly Arg Phe
Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Lys Lys Val Leu Thr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100
105 110 Thr Val Ser Ser 115 8 115 PRT Homo sapiens 8 Glu Val Gln
Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25
30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45 Ser Arg Ile Asn Gly Pro Gly Ala Thr Gly Tyr Ala Asp Ser
Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
Thr Leu Tyr Leu 65 70 75 80 Gln Ile Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys Ala 85 90 95 Lys His Gly Ala Pro Phe Asp Tyr
Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 9 116
PRT Homo sapiens 9 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val
Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Pro Ala Ser
Gly Leu His Thr Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe
Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Lys Pro Gly Leu Gly Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100
105 110 Thr Val Ser Ser 115 10 115 PRT Homo sapiens 10 Glu Val Gln
Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25
30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45 Ser Asp Ile Glu Arg Thr Gly Tyr Thr Arg Tyr Ala Asp Ser
Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys Ala 85 90 95 Lys Lys Val Leu Val Phe Asp Tyr
Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 11 116
PRT Homo sapiens 11 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val
Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Glu Ile Ser Ala Asn
Gly Ser Lys Thr Gln Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Leu
Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Lys Lys Val Leu Gln Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100
105 110 Thr Val Ser Ser 115 12 115 PRT Homo sapiens 12 Glu Val Gln
Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25
30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45 Ser Thr Ile Pro Ala Asn Gly Val Thr Arg Tyr Ala Asp Ser
Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys Ala 85 90 95 Lys Ser Leu Leu Gln Phe Asp Tyr
Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 13 116
PRT Homo sapiens 13 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val
Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Asp Ile Ala Ala Thr
Gly Ser Ala Thr Ser Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe
Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Lys Lys Ile Leu Lys Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100
105 110 Thr Val Ser Ser 115 14 116 PRT Homo sapiens 14 Glu Val Gln
Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Phe Ser Ser Tyr 20 25
30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45 Ser Thr Ile Ser Ser Val Gly Gln Ser Thr Arg Tyr Ala Asp
Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asn Leu Met Ser Phe Asp
Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 15
108 PRT Homo sapiens 15 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser
Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Asn 85 90
95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 16 108
PRT Homo sapiens 16 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser
Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Arg Ala Ser His Leu
Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly
Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp
Phe Ala Thr Tyr Tyr Cys Gln Gln Pro Trp Arg Ser Pro Gly 85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 17 108 PRT
Homo sapiens 17 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala
Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln
Ser Val Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Leu Ala Ser Arg Leu Gln
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe
Ala Thr Tyr Tyr Cys Gln Gln Asn Trp Trp Leu Pro Pro 85 90 95 Thr
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 18 107 PRT Homo
sapiens 18 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser
Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys
Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ser Leu Leu Gln Ser Gly
Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu 65 70 75 80 Asp Phe Ala Thr
Tyr Tyr Cys Gln Gln Arg Val Tyr Asp Pro Leu Thr 85 90 95 Phe Gly
Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 19 348 DNA Homo sapiens
19 gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc
cctgcgtctc 60 tcctgtgcag cctccggatt cacctttagc agctatgcca
tgagctgggt ccgccaggct 120 ccagggaagg gtctagagtg ggtctcagct
attagtggta gtggtggtag cacatactac 180 gcagactccg tgaagggccg
gttcaccatc tcccgtgaca attccaagaa cacgctgtat 240 ctgcaaatga
acagcctgcg tgccgaggac accgcggtat attactgtgc gaaaagttat 300
ggtgcttttg actactgggg ccagggaacc ctggtcaccg tctcgagc 348 20 348 DNA
Homo sapiens 20 gctcgagacg gtgaccaggg ttccctggcc ccagtagtca
aaagcaccat aacttttcgc 60 acagtaatat accgcggtgt cctcggcacg
caggctgttc atttgcagat acagcgtgtt 120 cttggaattg tcacgggaga
tggtgaaccg gcccttcacg gagtctgcgt agtatgtgct 180 accaccacta
ccactaatag ctgagaccca ctctagaccc ttccctggag cctggcggac 240
ccagctcatg gcatagctgc taaaggtgaa tccggaggct gcacaggaga gacgcaggga
300 ccccccaggc tgtaccaagc ctcccccaga ctccaacagc tgcacctc 348 21 120
PRT Homo sapiens VARIANT 103, 104, 105, 106 Xaa = Any Amino Acid 21
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5
10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser
Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr
Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ser Tyr Gly
Ala Xaa Xaa Xaa Xaa Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu
Val Thr Val Ser Ser 115 120 22 360 DNA Homo sapiens misc_feature
309, 312, 315, 318 K = G or T misc_feature 307, 308, 310, 311, 313,
314, 316, 317 n = A,T,C or G 22 gaggtgcagc tgttggagtc
tgggggaggc ttggtacagc ctggggggtc cctgcgtctc 60 tcctgtgcag
cctccggatt cacctttagc agctatgcca tgagctgggt ccgccaggct 120
ccagggaagg gtctagagtg ggtctcagct attagtggta gtggtggtag cacatactac
180 gcagactccg tgaagggccg gttcaccatc tcccgtgaca attccaagaa
cacgctgtat 240 ctgcaaatga acagcctgcg tgccgaggac accgcggtat
attactgtgc gaaaagttat 300 ggtgctnnkn nknnknnktt tgactactgg
ggccagggaa ccctggtcac cgtctcgagc 360 23 360 DNA Homo sapiens
misc_feature 43, 46, 49, 52 K = G or T misc_feature 44, 45, 47, 48,
50, 51, 53, 54 n = A,T,C or G 23 gctcgagacg gtgaccaggg ttccctggcc
ccagtagtca aaknnknnkn nknnagcacc 60 ataacttttc gcacagtaat
ataccgcggt gtcctcggca cgcaggctgt tcatttgcag 120 atacagcgtg
ttcttggaat tgtcacggga gatggtgaac cggcccttca cggagtctgc 180
gtagtatgtg ctaccaccac taccactaat agctgagacc cactctagac ccttccctgg
240 agcctggcgg acccagctca tggcatagct gctaaaggtg aatccggagg
ctgcacagga 300 gagacgcagg gaccccccag gctgtaccaa gcctccccca
gactccaaca gctgcacctc 360 24 324 DNA Homo sapiens 24 gacatccaga
tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga ccgtgtcacc 60
atcacttgcc gggcaagtca gagcattagc agctatttaa attggtacca gcagaaacca
120 gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg
ggtcccatca 180 cgtttcagtg gcagtggatc tgggacagat ttcactctca
ccatcagcag tctgcaacct 240 gaagattttg ctacgtacta ctgtcaacag
agttacagta cccctaatac gttcggccaa 300 gggaccaagg tggaaatcaa acgg 324
25 324 DNA Homo sapiens 25 ccgtttgatt tccaccttgg tcccttggcc
gaacgtatta ggggtactgt aactctgttg 60 acagtagtac gtagcaaaat
cttcaggttg cagactgctg atggtgagag tgaaatctgt 120 cccagatcca
ctgccactga aacgtgatgg gaccccactt tgcaaactgg atgcagcata 180
gatcaggagc ttaggggctt tccctggttt ctgctggtac caatttaaat agctgctaat
240 gctctgactt gcccggcaag tgatggtgac acggtctcct acagatgcag
acagggagga 300 tggagactgg gtcatctgga tgtc 324 26 324 DNA Homo
sapiens 26 gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga
ccgtgtcacc 60 atcacttgcc gggcaagtca gagcattatt aagcatttaa
agtggtacca gcagaaacca 120 gggaaagccc ctaagctcct gatctatggt
gcatcccggt tgcaaagtgg ggtcccatca 180 cgtttcagtg gcagtggatc
tgggacagat ttcactctca ccatcagcag tctgcaacct 240 gaagattttg
ctacgtacta ctgtcaacag ggggctcggt ggcctcagac gttcggccaa 300
gggaccaagg tggaaatcaa acgg 324 27 108 PRT Homo sapiens 27 Asp Ile
Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ile Lys His 20
25 30 Leu Lys Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu
Ile 35 40 45 Tyr Gly Ala Ser Arg Leu Gln Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln
Gln Gly Ala Arg Trp Pro Gln 85 90 95 Thr Phe Gly Gln Gly Thr Lys
Val Glu Ile Lys Arg 100 105 28 324 DNA Homo sapiens 28 gacatccaga
tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga ccgtgtcacc 60
atcacttgcc gggcaagtca gagcatttat tatcatttaa agtggtacca gcagaaacca
120 gggaaagccc ctaagctcct gatctataag gcatccacgt tgcaaagtgg
ggtcccatca 180 cgtttcagtg gcagtggatc tgggacagat ttcactctca
ccatcagcag tctgcaacct 240 gaagattttg ctacgtacta ctgtcaacag
gttcggaagg tgcctcggac gttcggccaa 300 gggaccaagg tggaaatcaa acgg 324
29 108 PRT Homo sapiens 29 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg
Ala Ser Gln Ser Ile Tyr Tyr His 20 25 30 Leu Lys Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Lys Ala Ser
Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Val Arg Lys Val Pro Arg 85
90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 30
360 DNA Homo sapiens 30 gaggtgcagc tgttggagtc tgggggaggc ttggtacagc
ctggggggtc cctgcgtctc 60 tcctgtgcag cctccggatt cacctttgag
tggtattgga tgggttgggt ccgccaggct 120 ccagggaagg gtctagagtg
ggtctcagct attagtggta gtggtggtag cacatactac 180 gcagactccg
tgaagggccg gttcaccatc tcccgcgaca attccaagaa cacgctgtat 240
ctgcaaatga acagcctgcg tgccgaggac accgcggtat attactgtgc gaaagttaag
300 ttgggggggg ggcctaattt tgactactgg ggccagggaa ccctggtcac
cgtctcgagc 360 31 120 PRT Homo sapiens 31 Glu Val Gln Leu Leu Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Arg Ile Ser Asp Glu 20 25 30 Asp Met
Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Ser Ile Tyr Gly Pro Ser Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50
55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Ser Ala Leu Glu Pro Leu Ser Glu Pro Leu
Gly Phe Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115
120 32 360 DNA Homo sapiens 32 gaggtgcagc tgttggagtc tgggggaggc
ttggtacagc ctggggggtc cctgcgtctc 60 tcctgtgcag cctccggatt
taggattagc gatgaggata tgggctgggt ccgccaggct 120 ccagggaagg
gtctagagtg ggtatcaagc atttatggcc ctagcggtag cacatactac 180
gcagactccg tgaagggccg gttcaccatc tcccgtgaca attccaagaa cacgctgtat
240 ctgcaaatga acagcctgcg tgccgaggac accgcggtat attattgcgc
gagtgctttg 300 gagccgcttt cggagcccct gggcttttgg ggtcagggaa
ccctggtcac cgtctcgagc 360 33 116 PRT Homo sapiens 33 Glu Val Gln
Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Leu Tyr 20 25
30 Asn Met Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45 Ser Phe Ile Ser Gln Thr Gly Arg Leu Thr Trp Tyr Ala Asp
Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Thr Leu Glu Asp Phe Asp
Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 34
348 DNA Homo sapiens 34 gaggtgcagc tgttggagtc tgggggaggc ttggtacagc
ctggggggtc cctgcgtctc 60 tcctgtgcag cctccggatt cacctttgat
ctttataata tgttttgggt ccgccaggct 120 ccagggaagg gtctagagtg
ggtctcattt attagtcaga ctggtaggct tacatggtac 180 gcagactccg
tgaagggccg gttcaccatc tcccgcgaca attccaagaa cacgctgtat 240
ctgcaaatga acagcctgcg tgccgaggac accgcggtat attactgtgc gaaaacgctg
300 gaggattttg actactgggg ccagggaacc ctggtcaccg tctcgagc 348 35 108
PRT Homo sapiens 35 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser
Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
Gln Ser Val Lys Glu Phe 20 25 30 Leu Trp Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Met Ala Ser Asn Leu
Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly
Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp
Phe Ala Thr Tyr Tyr Cys Gln Gln Lys Phe Lys Leu Pro Arg 85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 36 324 DNA
Homo sapiens 36 gacatccaga tgacccagtc tccatcctcc ctgtctgcat
ctgtaggaga ccgtgtcacc 60 atcacttgcc gggcaagtca gagcgttaag
gagtttttat ggtggtacca gcagaaacca 120 gggaaagccc ctaagctcct
gatctatatg gcatccaatt tgcaaagtgg ggtcccatca 180 cgtttcagtg
gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240
gaagattttg ctacgtacta ctgtcaacag aagtttaagc tgcctcgtac gttcggccaa
300 gggaccaagg tggaaatcaa acgg 324 37 108 PRT Homo sapiens 37 Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10
15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Asp Ser Tyr
20 25 30 Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45 Tyr Ser Ala Ser Glu Leu Gln Ser Gly Val Pro Ser
Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln Val Val Trp Arg Pro Phe 85 90 95 Thr Phe Gly Gln Gly Thr
Lys Val Glu Ile Lys Arg 100 105 38 324 DNA Homo sapiens 38
gacatccaga tgacccagtc tccatcctct ctgtctgcat ctgtaggaga ccgtgtcacc
60 atcacttgcc gggcaagtca gagcattgat agttatttac attggtacca
gcagaaacca 120 gggaaagccc ctaagctcct gatctatagt gcatccgagt
tgcaaagtgg ggtcccatca 180 cgtttcagtg gcagtggatc tgggacagat
ttcactctca ccatcagcag tctgcaacct 240 gaagattttg ctacgtacta
ctgtcaacag gttgtgtggc gtccttttac gttcggccaa 300 gggaccaagg
tggaaatcaa acgc 324 39 324 DNA Homo sapiens 39 gcgtttgatt
tccaccttgg tcccttggcc gaacgtaaaa ggacgccaca caacctgttg 60
acagtagtac gtagcaaaat cttcaggttg cagactgctg atggtgagag tgaaatctgt
120 cccagatcca ctgccactga aacgtgatgg gaccccactt tgcaactcgg
atgcactata 180 gatcaggagc ttaggggctt tccctggttt ctgctggtac
caatgtaaat aactatcaat 240 gctctgactt gcccggcaag tgatggtgac
acggtctcct acagatgcag acagagagga 300 tggagactgg gtcatctgga tgtc 324
40 108 PRT Homo sapiens 40 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg
Ala Ser Gln Ser Ile Phe Met Asn 20 25 30 Leu Leu Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asn Ala Ser
Val Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Val Val Trp Arg Pro Phe 85
90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 41
324 DNA Homo sapiens 41 gacatccaga tgacccagtc tccatcctcc ctgtctgcat
ctgtaggaga ccgtgtcacc 60 atcacttgcc gggcaagtca gagcattttt
atgaatttat tgtggtacca gcagaaacca 120 gggaaagccc ctaagctcct
gatctataat gcatccgtgt tgcaaagtgg ggtcccatca 180 cgtttcagtg
gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240
gaagattttg ctacgtacta ctgtcaacag gttgtgtggc gtccttttac gttcggccaa
300 gggaccaagg tggaaatcaa acgg 324 42 324 DNA Homo sapiens 42
ccgtttgatt tccaccttgg tcccttggcc gaacgtaaaa ggacgccaca caacctgttg
60 acagtagtac gtagcaaaat cttcaggttg cagactgctg atggtgagag
tgaaatctgt 120 cccagatcca ctgccactga aacgtgatgg gaccccactt
tgcaacacgg atgcattata 180 gatcaggagc ttaggggctt tccctggttt
ctgctggtac cacaataaat tcataaaaat 240 gctctgactt gcccggcaag
tgatggtgac acggtctcct acagatgcag acagggagga 300 tggagactgg
gtcatctgga tgtc 324 43 108 PRT Homo sapiens 43 Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val
Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Tyr Asp Ala 20 25 30 Leu
Glu Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40
45 Tyr Thr Ala Ser Arg Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Val Met
Gln Arg Pro Val 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys Arg 100 105 44 324 DNA Homo sapiens 44 gacatccaga tgacccagtc
tccatcctcc ctgtctgcat ctgtaggaga ccgtgtcacc 60 atcacttgcc
gggcaagtca gagcatttat gatgcgttag agtggtacca gcagaaacca 120
gggaaagccc ctaagctcct gatctatact gcatcccggt tgcaaagtgg ggtcccatca
180 cgtttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag
tctgcaacct 240 gaagattttg ctacgtacta ctgtcaacag gttatgcagc
gtcctgttac gttcggccaa 300 gggaccaagg tggaaatcaa acgg 324 45 324 DNA
Homo sapiens 45 ccgtttgatt tccaccttgg tcccttggcc gaacgtaaca
ggacgctgca taacctgttg 60 acagtagtac gtagcaaaat cttcaggttg
cagactgctg atggtgagag tgaaatctgt 120 cccagatcca ctgccactga
aacgtgatgg gaccccactt tgcaaccggg atgcagtata 180 gatcaggagc
ttaggggctt tccctggttt ctgctggtac cactctaacg catcataaat 240
gctctgactt gcccggcaag tgatggtgac acggtctcct acagatgcag acagggagga
300 tggagactgg gtcatctgga tgtc 324 46 107 PRT Homo sapiens 46 Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10
15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Tyr Asp Ala
20 25 30 Leu Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu
Ile Tyr 35 40 45 Thr Ala Ser Arg Leu Gln Ser Gly Val Pro Ser Arg
Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
Ser Ser Leu Gln Pro Glu 65 70 75 80 Asp Phe Ala Thr Tyr His Cys Gln
Gln Val Met Gln Arg Pro Val Thr 85 90 95 Phe Gly Gln Gly Thr Lys
Val Glu Ile Lys Arg 100 105 47 324 DNA Homo sapiens 47 gacatccaga
tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga ccgtgtcacc 60
atcacttgcc gggcaagtca gagcatttat gatgctttac agtggtacca gcagaaacca
120 gggaaagccc ctaagctcct gatctatact gcatcccggt tgcaaagtgg
ggtcccatca 180 cgtttcagtg gcagtggatc tgggacagat ttcactctca
ccatcagcag tctgcaacct 240 gaagattttg ctacgtacca ctgtcaacag
gttatgcagc gtcctgttac gttcggccaa 300 gggaccaagg tggaaatcaa acgg 324
48 324 DNA Homo sapiens 48 ccgtttgatt tccaccttgg tcccttggcc
gaacgtaaca ggacgctgca taacctgttg 60 acagtggtac gtagcaaaat
cttcaggttg cagactgctg atggtgagag tgaaatctgt 120 cccagatcca
ctgccactga aacgtgatgg gaccccactt tgcaaccggg atgcagtata 180
gatcaggagc ttaggggctt tccctggttt ctgctggtac cactgtaaag catcataaat
240 gctctgactt gcccggcaag tgatggtgac acggtctcct acagatgcag
acagggagga 300 tggagactgg gtcatctgga tgtc 324 49 108 PRT Homo
sapiens 49 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser
Val Lys Glu Phe 20 25 30 Leu Trp Trp Tyr Gln Gln Lys Pro Gly Lys
Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Met Ala Ser Asn Leu Gln Ser
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Lys Phe Lys Leu Pro Arg 85 90 95 Thr Phe
Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 50 324 DNA Homo
sapiens 50 gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga
ccgtgtcacc 60 atcacttgcc gggcaagtca gagcgttaag gagtttttat
ggtggtacca gcagaaacca 120 gggaaagccc ctaagctcct gatctatatg
gcatccaatt tgcaaagtgg ggtcccatca 180 cgtttcagtg gcagtggatc
tgggacagat ttcactctca ccatcagcag tctgcaacct 240 gaagattttg
ctacgtacta ctgtcaacag aagtttaagc tgcctcgtac gttcggccaa 300
gggaccaagg tggaaatcaa acgg 324 51 324 DNA Homo sapiens 51
ccgtttgatt tccaccttgg tcccttggcc gaacgtacga ggcagcttaa acttctgttg
60 acagtagtac gtagcaaaat cttcaggttg cagactgctg atggtgagag
tgaaatctgt 120 cccagatcca ctgccactga aacgtgatgg gaccccactt
tgcaaattgg atgccatata 180 gatcaggagc ttaggggctt tccctggttt
ctgctggtac caccataaaa actccttaac 240 gctctgactt gcccggcaag
tgatggtgac acggtctcct acagatgcag acagggagga 300 tggagactgg
gtcatctgga tgtc 324 52 108 PRT Homo sapiens 52 Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val
Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Trp Thr Lys 20 25 30 Leu
His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35
40
45 Tyr Met Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Phe
Ser Asn Pro Ser 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys Arg 100 105 53 324 DNA Homo sapiens 53 gacatccaga tgacccagtc
tccatcctcc ctgtctgcat ctgtaggaga ccgtgtcacc 60 atcacttgcc
gggcaagtca gagcatttgg acgaagttac attggtacca gcagaaacca 120
gggaaagccc ctaagctcct gatctatatg gcatccagtt tgcaaagtgg ggtcccatca
180 cgtttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag
tctgcaacct 240 gaagattttg ctacgtacta ctgtcaacag tggtttagta
atcctagtac gttcggccaa 300 gggaccaagg tggaaatcaa acgc 324 54 324 DNA
Homo sapiens 54 gcgtttgatt tccaccttgg tcccttggcc gaacgtacta
ggattactaa accactgttg 60 acagtagtac gtagcaaaat cttcaggttg
cagactgctg atggtgagag tgaaatctgt 120 cccagatcca ctgccactga
aacgtgatgg gaccccactt tgcaaactgg atgccatata 180 gatcaggagc
ttaggggctt tccctggttt ctgctggtac caatgtaact tcgtccaaat 240
gctctgactt gcccggcaag tgatggtgac acggtctcct acagatgcag acagggagga
300 tggagactgg gtcatctgga tgtc 324 55 107 PRT Homo sapiens 55 Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10
15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Pro Ile Leu
20 25 30 Cys Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu
Ile Tyr 35 40 45 Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg
Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
Ser Ser Leu Gln Pro Glu 65 70 75 80 Asp Phe Ala Thr Tyr Tyr Cys Gln
Gln Ile Gln His Ile Pro Val Thr 85 90 95 Phe Gly Gln Gly Thr Lys
Val Glu Ile Lys Arg 100 105 56 324 DNA Homo sapiens 56 gacatccaga
tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga ccgtgtcacc 60
atcacttgcc gggcaagtca gagcatttag ccgattttat gttggtacca gcagaaacca
120 gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg
ggtcccatca 180 cgtttcagtg gcagtggatc tgggacagat ttcactctca
ccatcagcag tctgcaacct 240 gaagattttg ctacgtacta ctgtcaacag
attcagcata ttcctgtgac gttcggccaa 300 gggaccaagg tggaaatcaa acgg 324
57 324 DNA Homo sapiens 57 ccgtttgatt tccaccttgg tcccttggcc
gaacgtcaca ggaatatgct gaatctgttg 60 acagtagtac gtagcaaaat
cttcaggttg cagactgctg atggtgagag tgaaatctgt 120 cccagatcca
ctgccactga aacgtgatgg gaccccactt tgcaaactgg atgcagcata 180
gatcaggagc ttaggggctt tccctggttt ctgctggtac caacataaaa tcggctaaat
240 gctctgactt gcccggcaag tgatggtgac acggtctcct acagatgcag
acagggagga 300 tggagactgg gtcatctgga tgtc 324 58 107 PRT Homo
sapiens 58 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser
Ile Gly Asp Leu 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Lys Ala
Pro Lys Leu Leu Ile Tyr 35 40 45 Thr Ala Ser Leu Leu Gln Ser Gly
Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu 65 70 75 80 Asp Phe Ala Thr
Tyr Tyr Cys Gln Gln Gln Ser Ala Phe Pro Asn Thr 85 90 95 Leu Gly
Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 59 324 DNA Homo sapiens
59 gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga
ccgtgtcacc 60 atcacttgcc gggcaagtca gagcattggg taggatttac
attggtacca gcagaaacca 120 gggaaagccc ctaagctcct gatctatacg
gcatcccttt tgcaaagtgg ggtcccatca 180 cgtttcagtg gcagtggatc
tgggacagat ttcactctca ccatcagcag tctgcaacct 240 gaagattttg
ctacgtacta ctgtcaacag cagagtgctt ttcctaatac gctcggccaa 300
gggaccaagg tggaaatcaa acgg 324 60 324 DNA Homo sapiens 60
ccgtttgatt tccaccttgg tcccttggcc gagcgtatta ggaaaagcac tctgctgttg
60 acagtagtac gtagcaaaat cttcaggttg cagactgctg atggtgagag
tgaaatctgt 120 cccagatcca ctgccactga aacgtgatgg gaccccactt
tgcaaaaggg atgccgtata 180 gatcaggagc ttaggggctt tccctggttt
ctgctggtac caatgtaaat cctacccaat 240 gctctgactt gcccggcaag
tgatggtgac acggtctcct acagatgcag acagggagga 300 tggagactgg
gtcatctgga tgtc 324 61 107 PRT Homo sapiens 61 Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val
Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Thr Lys Asn 20 25 30 Leu
Leu Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40
45 Tyr Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser
50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln
Pro Glu 65 70 75 80 Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Leu Arg His
Lys Pro Pro Thr 85 90 95 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
Arg 100 105 62 324 DNA Homo sapiens 62 gacatccaga tgacccagtc
tccatcctcc ctgtctgcat ccgtaggaga ccgtgtcacc 60 atcacttgcc
gggcaagtca gagcataacg aagaatttac tttggtacca gcagaaacca 120
gggaaagccc ctaagctcct gatctattag gcatcctctt tgcaaagtgg ggtcccatca
180 cgtttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag
tctgcaacct 240 gaagattttg ctacgtacta ctgtcaacag cttcgtcata
agcctccgac gttcggccaa 300 gggaccaagg tggaaatcaa acgg 324 63 324 DNA
Homo sapiens 63 ccgtttgatt tccaccttgg tcccttggcc gaacgtcgga
ggcttatgac gaagctgttg 60 acagtagtac gtagcaaaat cttcaggttg
cagactgctg atggtgagag tgaaatctgt 120 cccagatcca ctgccactga
aacgtgatgg gaccccactt tgcaaagagg atgcctaata 180 gatcaggagc
ttaggggctt tccctggttt ctgctggtac caaagtaaat tcttcgttat 240
gctctgactt gcccggcaag tgatggtgac acggtctcct acggatgcag acagggagga
300 tggagactgg gtcatctgga tgtc 324 64 107 PRT Homo sapiens 64 Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10
15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Lys Ser Leu
20 25 30 Arg Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu
Ile Tyr 35 40 45 His Ala Ser Asp Leu Gln Ser Gly Val Pro Ser Arg
Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
Ser Ser Leu Gln Pro Glu 65 70 75 80 Asp Phe Ala Thr Tyr Tyr Cys Gln
Gln Met Val Asn Ser Pro Val Thr 85 90 95 Phe Gly Gln Gly Thr Lys
Val Glu Ile Lys Arg 100 105 65 324 DNA Homo sapiens 65 gacatccaga
tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga ccgtgtcacc 60
atcacttgcc gggcaagtca gagcatttag aagtctttaa ggtggtacca gcagaaacca
120 gggaaagccc ctaagctcct gatctatcat gcatccgatt tgcaaagtgg
ggtcccatca 180 cgtttcagtg gcagtggatc tgggacagat ttcactctca
ccatcagcag tctgcaacct 240 gaagattttg ctacgtacta ctgtcaacag
atggttaata gtcctgttac gttcggccaa 300 gggaccaagg tggaaatcaa acgg 324
66 324 DNA Homo sapiens 66 ccgtttgatt tccaccttgg tcccttggcc
gaacgtaaca ggactattaa ccatctgttg 60 acagtagtac gtagcaaaat
cttcaggttg cagactgctg atggtgagag tgaaatctgt 120 cccagatcca
ctgccactga aacgtgatgg gaccccactt tgcaaatcgg atgcatgata 180
gatcaggagc ttaggggctt tccctggttt ctgctggtac caccttaaag acttctaaat
240 gctctgactt gcccggcaag tgatggtgac acggtctcct acagatgcag
acagggagga 300 tggagactgg gtcatctgga tgtc 324 67 107 PRT Homo
sapiens 67 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser
Ile Thr Ala Leu 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Lys Ala
Pro Lys Leu Leu Ile Tyr 35 40 45 Ser Ala Ser Ser Leu Gln Ser Gly
Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu 65 70 75 80 Asp Phe Ala Thr
Tyr Tyr Cys Gln Gln Ser Ser Phe Leu Pro Phe Thr 85 90 95 Phe Gly
Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 68 324 DNA Homo sapiens
68 gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga
ccgtgtcacc 60 atcacttgcc gggcaagtca gagcatttag acggcgttac
attggtacca gcagaaacca 120 gggaaagccc ctaagctcct gatctattct
gcatccagtt tgcaaagtgg ggtcccatca 180 cgtttcagtg gcagtggatc
tgggacagat ttcactctca ccatcagcag tctgcaacct 240 gaagattttg
ctacgtacta ctgtcaacag tcgagttttt tgccttttac gttcggccaa 300
gggaccaagg tggaaatcaa acgg 324 69 324 DNA Homo sapiens 69
ccgtttgatt tccaccttgg tcccttggcc gaacgtaaaa ggcaaaaaac tcgactgttg
60 acagtagtac gtagcaaaat cttcaggttg cagactgctg atggtgagag
tgaaatctgt 120 cccagatcca ctgccactga aacgtgatgg gaccccactt
tgcaaactgg atgcagaata 180 gatcaggagc ttaggggctt tccctggttt
ctgctggtac caatgtaacg ccgtctaaat 240 gctctgactt gcccggcaag
tgatggtgac acggtctcct acagatgcag acagggagga 300 tggagactgg
gtcatctgga tgtc 324 70 108 PRT Homo sapiens 70 Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val
Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Pro Asn 20 25 30 Leu
Glu Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40
45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gln Met
Gly Arg Pro Arg 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys Arg 100 105 71 324 DNA Homo sapiens 71 gacatccaga tgacccagtc
tccatcctcc ctgtctgcat ctgtaggaga ccgtgtcacc 60 atcacttgcc
gggcaagtca gagcattggg ccgaatttag agtggtacca gcagaaacca 120
gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca
180 cgtttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag
tctgcaacct 240 gaagattttg ctacgtacta ctgtcaacag cagatggggc
gtcctcggac gttcggccaa 300 gggaccaagg tggaaatcaa acgg 324 72 324 DNA
Homo sapiens 72 ccgtttgatt tccaccttgg tcccttggcc gaacgtccga
ggacgcccca tctgctgttg 60 acagtagtac gtagcaaaat cttcaggttg
cagactgctg atggtgagag tgaaatctgt 120 cccagatcca ctgccactga
aacgtgatgg gaccccactt tgcaaactgg atgcagcata 180 gatcaggagc
ttaggggctt tccctggttt ctgctggtac cactctaaat tcggcccaat 240
gctctgactt gcccggcaag tgatggtgac acggtctcct acagatgcag acagggagga
300 tggagactgg gtcatctgga tgtc 324 73 107 PRT Homo sapiens 73 Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10
15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Lys His Leu
20 25 30 Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu
Ile Tyr 35 40 45 Lys Ala Ser Val Leu Gln Ser Gly Val Pro Ser Arg
Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
Ser Ser Leu Gln Pro Glu 65 70 75 80 Asp Phe Ala Thr Tyr Tyr Cys Gln
Gln Leu Arg Arg Arg Pro Thr Thr 85 90 95 Phe Gly Gln Gly Thr Lys
Val Glu Ile Lys Arg 100 105 74 324 DNA Homo sapiens 74 gacatccaga
tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga ccgtgtcacc 60
atcacttgcc gggcaagtca gagcattaag cattagttag cttggtacca gcagaaacca
120 gggaaagccc ctaagctcct gatctataag gcatccgtgt tgcaaagtgg
ggtcccatca 180 cgtttcagtg gcagtggatc tgggacagat ttcactctca
ccatcagcag tctgcaacct 240 gaagattttg ctacgtacta ctgtcaacag
cttaggcgtc gtcctactac gttcggccaa 300 gggaccaagg tggaaatcaa acgg 324
75 324 DNA Homo sapiens 75 ccgtttgatt tccaccttgg tcccttggcc
gaacgtagta ggacgacgcc taagctgttg 60 acagtagtac gtagcaaaat
cttcaggttg cagactgctg atggtgagag tgaaatctgt 120 cccagatcca
ctgccactga aacgtgatgg gaccccactt tgcaacacgg atgccttata 180
gatcaggagc ttaggggctt tccctggttt ctgctggtac caagctaact aatgcttaat
240 gctctgactt gcccggcaag tgatggtgac acggtctcct acagatgcag
acagggagga 300 tggagactgg gtcatctgga tgtc 324 76 107 PRT Homo
sapiens 76 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser
Val Lys Ala Leu 20 25 30 Thr Trp Tyr Gln Gln Lys Pro Gly Lys Ala
Pro Lys Leu Leu Ile Tyr 35 40 45 Lys Ala Ser Thr Leu Gln Ser Gly
Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu 65 70 75 80 Asp Phe Ala Thr
Tyr Tyr Cys Gln Gln His Ser Ser Arg Pro Tyr Thr 85 90 95 Phe Gly
Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 77 324 DNA Homo sapiens
77 gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga
ccgtgtcacc 60 atcacttgcc gggcaagtca gagcgttaag gcttagttaa
cttggtacca gcagaaacca 120 gggaaagccc ctaagctcct gatctataag
gcatccactt tgcaaagtgg ggtcccatca 180 cgtttcagtg gcagtggatc
tgggacagat ttcactctca ccatcagcag tctgcaacct 240 gaagattttg
ctacgtacta ctgtcaacag catagttcta ggccttatac gttcggccaa 300
gggaccaagg tggaaatcaa acgg 324 78 324 DNA Homo sapiens 78
ccgtttgatt tccaccttgg tcccttggcc gaacgtataa ggcctagaac tatgctgttg
60 acagtagtac gtagcaaaat cttcaggttg cagactgctg atggtgagag
tgaaatctgt 120 cccagatcca ctgccactga aacgtgatgg gaccccactt
tgcaaagtgg atgccttata 180 gatcaggagc ttaggggctt tccctggttt
ctgctggtac caagttaact aagccttaac 240 gctctgactt gcccggcaag
tgatggtgac acggtctcct acagatgcag acagggagga 300 tggagactgg
gtcatctgga tgtc 324 79 107 PRT Homo sapiens 79 Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val
Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Glu Asn Arg 20 25 30 Leu
Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40
45 Tyr Ala Ser Leu Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser
50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln
Pro Glu 65 70 75 80 Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Asp Ser Tyr
Phe Pro Arg Thr 85 90 95 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
Arg 100 105 80 324 DNA Homo sapiens 80 gacatccaga tgacccagtc
tccatcctcc ctgtctgcat ctgtaggaga ccgtgtcacc 60 atcacttgcc
gggcaagtca gagcattgag aatcggttag gttggtacca gcagaaacca 120
gggaaagccc ctaagctcct gatctattag gcgtccttgt tgcaaagtgg ggtcccatca
180 cgtttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag
tctgcaacct 240 gaagattttg ctacgtacta ctgtcaacag gattcgtatt
ttcctcgtac gttcggccaa 300 gggaccaagg tggaaatcaa acgg 324 81 324 DNA
Homo sapiens 81 ccgtttgatt tccaccttgg tcccttggcc gaacgtacga
ggaaaatacg aatcctgttg 60 acagtagtac gtagcaaaat cttcaggttg
cagactgctg atggtgagag tgaaatctgt 120 cccagatcca ctgccactga
aacgtgatgg gaccccactt tgcaacaagg acgcctaata 180 gatcaggagc
ttaggggctt tccctggttt ctgctggtac caacctaacc gattctcaat 240
gctctgactt gcccggcaag tgatggtgac acggtctcct acagatgcag acagggagga
300 tggagactgg gtcatctgga tgtc 324 82 107 PRT Homo sapiens 82 Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10
15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Met Asp Lys
20 25 30 Leu Lys Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45 Tyr Ala Ser Ile Leu Gln Ser Gly Val Pro Ser Arg
Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
Ser Ser Leu Gln Pro Glu 65 70 75 80 Asp Phe Ala Thr Tyr Tyr Cys Gln
Gln Asp Ser Gly Gly Pro Asn Thr 85 90 95 Phe Gly Gln Gly Thr Lys
Val Glu Ile Lys Arg 100 105 83 324 DNA Homo sapiens 83 gacatccaga
tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga
ccgtgtcacc 60 atcacttgcc gggcaagtca gagcattatg gataagttaa
agtggtacca gcagaaacca 120 gggaaagccc ctaagctcct gatctattag
gcatccattt tgcaaagtgg ggtcccatca 180 cgtttcagtg gcagtggatc
tgggacagat ttcactctca ccatcagcag tctgcaacct 240 gaagattttg
ctacgtacta ctgtcaacag gatagtgggg gtcctaatac gttcggccaa 300
gggaccaagg tggaaatcaa acgg 324 84 324 DNA Homo sapiens 84
ccgtttgatt tccaccttgg tcccttggcc gaacgtatta ggacccccac tatcctgttg
60 acagtagtac gtagcaaaat cttcaggttg cagactgctg atggtgagag
tgaaatctgt 120 cccagatcca ctgccactga aacgtgatgg gaccccactt
tgcaaaatgg atgcctaata 180 gatcaggagc ttaggggctt tccctggttt
ctgctggtac cactttaact tatccataat 240 gctctgactt gcccggcaag
tgatggtgac acggtctcct acagatgcag acagggagga 300 tggagactgg
gtcatctgga tgtc 324 85 108 PRT Homo sapiens 85 Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val
Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Arg Asn 20 25 30 Leu
Glu Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40
45 Tyr Asp Ala Ser His Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Arg
Trp Leu Pro Arg 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys Arg 100 105 86 324 DNA Homo sapiens 86 gacatccaga tgacccagtc
tccatcctcc ctgtctgcat ctgtaggaga ccgtgtcacc 60 atcacttgcc
gggcaagtca gagcattggg aggaatttag agtggtacca gcagaaacca 120
gggaaagccc ctaagctcct gatctatgat gcatcccatt tgcaaagtgg ggtcccatca
180 cgtttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag
tctgcaacct 240 gaagattttg ctacgtacta ctgtcaacag tcgcgttggc
ttcctcgtac gttcggccaa 300 gggaccaagg tggaaatcaa acgg 324 87 324 DNA
Homo sapiens 87 ccgtttgatt tccaccttgg tcccttggcc gaacgtacga
ggaagccaac gcgactgttg 60 acagtagtac gtagcaaaat cttcaggttg
cagactgctg atggtgagag tgaaatctgt 120 cccagatcca ctgccactga
aacgtgatgg gaccccactt tgcaaatggg atgcatcata 180 gatcaggagc
ttaggggctt tccctggttt ctgctggtac cactctaaat tcctcccaat 240
gctctgactt gcccggcaag tgatggtgac acggtctcct acagatgcag acagggagga
300 tggagactgg gtcatctgga tgtc 324 88 108 PRT Homo sapiens 88 Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10
15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Arg Lys Met
20 25 30 Leu Val Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45 Tyr Arg Ala Ser Tyr Leu Gln Ser Gly Val Pro Ser
Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln Ala Phe Arg Arg Pro Arg 85 90 95 Thr Phe Gly Gln Gly Thr
Lys Val Glu Ile Lys Arg 100 105 89 324 DNA Homo sapiens 89
gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga ccgtgtcacc
60 atcacttgcc gggcaagtca gagcattagg aagatgttag tttggtacca
gcagaaacca 120 gggaaagccc ctaagctcct gatctatcgg gcatcctatt
tgcaaagtgg ggtcccatca 180 cgtttcagtg gcagtggatc tgggacagat
ttcactctca ccatcagcag tctgcaacct 240 gaagattttg ctacgtacta
ctgtcaacag gcttttcggc ggcctaggac gttcggccaa 300 gggaccaagg
tggaaatcaa acgg 324 90 324 DNA Homo sapiens 90 ccgtttgatt
tccaccttgg tcccttggcc gaacgtccta ggccgccgaa aagcctgttg 60
acagtagtac gtagcaaaat cttcaggttg cagactgctg atggtgagag tgaaatctgt
120 cccagatcca ctgccactga aacgtgatgg gaccccactt tgcaaatagg
atgcccgata 180 gatcaggagc ttaggggctt tccctggttt ctgctggtac
caaactaaca tcttcctaat 240 gctctgactt gcccggcaag tgatggtgac
acggtctcct acagatgcag acagggagga 300 tggagactgg gtcatctgga tgtc 324
91 115 PRT Homo sapiens 91 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Asp Leu Tyr 20 25 30 Asn Met Phe Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Phe Ile Ser
Gln Thr Gly Arg Leu Thr Trp Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Lys Thr Leu Glu Asp Phe Asp Tyr Trp Gly Gln Gly Thr Leu
Val 100 105 110 Thr Val Ser 115 92 345 DNA Homo sapiens 92
gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgcgtctc
60 tcctgtgcag cctccggatt cacctttgat ctttataata tgttttgggt
ccgccaggct 120 ccagggaagg gtctagagtg ggtctcattt attagtcaga
ctggtaggct tacatggtac 180 gcagactccg tgaagggccg gttcaccatc
tcccgcgaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgcg
tgccgaggac accgcggtat attactgtgc gaaaacgctg 300 gaggattttg
actactgggg ccagggaacc ctggtcaccg tctcg 345 93 345 DNA Homo sapiens
93 cgagacggtg accagggttc cctggcccca gtagtcaaaa tcctccagcg
ttttcgcaca 60 gtaatatacc gcggtgtcct cggcacgcag gctgttcatt
tgcagataca gcgtgttctt 120 ggaattgtcg cgggagatgg tgaaccggcc
cttcacggag tctgcgtacc atgtaagcct 180 accagtctga ctaataaatg
agacccactc tagacccttc cctggagcct ggcggaccca 240 aaacatatta
taaagatcaa aggtgaatcc ggaggctgca caggagagac gcagggaccc 300
cccaggctgt accaagcctc ccccagactc caacagctgc acctc 345 94 119 PRT
Homo sapiens 94 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Thr Phe Pro Val Tyr 20 25 30 Met Met Gly Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Asp Ala Leu Gly
Gly Arg Thr Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala
Lys Thr Met Ser Asn Lys Thr His Thr Phe Asp Tyr Trp Gly Gln 100 105
110 Gly Thr Leu Val Thr Val Ser 115 95 357 DNA Homo sapiens 95
gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgcgtctc
60 tcctgtgcag cctccggatt cacctttccg gtttatatga tgggttgggt
ccgccaggct 120 ccagggaagg gtctagagtg ggtctcatcg attgatgctc
ttggtgggcg gacaggttac 180 gcagactccg tgaagggccg gttcaccatc
tcccgcgaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgcg
tgccgaggac accgcggtat attactgtgc gaaaactatg 300 tcgaataaga
cgcatacgtt tgactactgg ggccagggaa ccctggtcac cgtctcg 357 96 357 DNA
Homo sapiens 96 cgagacggtg accagggttc cctggcccca gtagtcaaac
gtatgcgtct tattcgacat 60 agttttcgca cagtaatata ccgcggtgtc
ctcggcacgc aggctgttca tttgcagata 120 cagcgtgttc ttggaattgt
cgcgggagat ggtgaaccgg cccttcacgg agtctgcgta 180 acctgtccgc
ccaccaagag catcaatcga tgagacccac tctagaccct tccctggagc 240
ctggcggacc caacccatca tataaaccgg aaaggtgaat ccggaggctg cacaggagag
300 acgcagggac cccccaggct gtaccaagcc tcccccagac tccaacagct gcacctc
357 97 114 PRT Homo sapiens 97 Glu Val Gln Leu Leu Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Val Ala Tyr 20 25 30 Asn Met Thr Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile
Asn Thr Phe Gly Asn Thr Arg Tyr Ala Asp Ser Val Lys 50 55 60 Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70
75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
Ala 85 90 95 Lys Gly Ser Arg Pro Phe Asp Tyr Trp Gly Gln Gly Thr
Leu Val Thr 100 105 110 Val Ser 98 345 DNA Homo sapiens 98
gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgcgtctc
60 tcctgtgcag cctccggatt cacctttgtg gcttataata tgacttgggt
ccgccaggct 120 ccagggaagg gtctagagtg ggtctcaagt attaatactt
ttggtaatta gacaaggtac 180 gcagactccg tgaagggccg gttcaccatc
tcccgcgaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgcg
tgccgaggac accgcggtat attactgtgc gaaaggtagt 300 aggccttttg
actactgggg ccagggaacc ctggtcaccg tctcg 345 99 345 DNA Homo sapiens
99 cgagacggtg accagggttc cctggcccca gtagtcaaaa ggcctactac
ctttcgcaca 60 gtaatatacc gcggtgtcct cggcacgcag gctgttcatt
tgcagataca gcgtgttctt 120 ggaattgtcg cgggagatgg tgaaccggcc
cttcacggag tctgcgtacc ttgtctaatt 180 accaaaagta ttaatacttg
agacccactc tagacccttc cctggagcct ggcggaccca 240 agtcatatta
taagccacaa aggtgaatcc ggaggctgca caggagagac gcagggaccc 300
cccaggctgt accaagcctc ccccagactc caacagctgc acctc 345 100 118 PRT
Homo sapiens 100 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val
Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Gly Tyr Arg 20 25 30 Met Gly Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val Ser 35 40 45 Trp Ile Thr Arg Thr Gly
Gly Thr Thr Gln Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95
Lys Pro Ala Lys Leu Val Gly Val Gly Phe Asp Tyr Trp Gly Gln Gly 100
105 110 Thr Leu Val Thr Val Ser 115 101 357 DNA Homo sapiens 101
gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgcgtctc
60 tcctgtgcag cctccggatt caccttttag gggtatcgta tgggttgggt
ccgccaggct 120 ccagggaagg gtctagagtg ggtctcatgg attacgcgta
ctggtgggac gacacagtac 180 gcagactccg tgaagggccg gttcaccatc
tcccgcgaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgcg
tgccgaggac accgcggtat attactgtgc gaaaccggcg 300 aagcttgttg
gggttgggtt tgactactgg ggccagggaa ccctggtcac cgtctcg 357 102 357 DNA
Homo sapiens 102 cgagacggtg accagggttc cctggcccca gtagtcaaac
ccaaccccaa caagcttcgc 60 cggtttcgca cagtaatata ccgcggtgtc
ctcggcacgc aggctgttca tttgcagata 120 cagcgtgttc ttggaattgt
cgcgggagat ggtgaaccgg cccttcacgg agtctgcgta 180 ctgtgtcgtc
ccaccagtac gcgtaatcca tgagacccac tctagaccct tccctggagc 240
ctggcggacc caacccatac gataccccta aaaggtgaat ccggaggctg cacaggagag
300 acgcagggac cccccaggct gtaccaagcc tcccccagac tccaacagct gcacctc
357 103 118 PRT Homo sapiens 103 Glu Val Gln Leu Leu Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Arg Lys Tyr 20 25 30 Met Gly Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser 35 40 45 Gln Ile
Gly Ala Lys Gly Gln Ser Thr Asp Tyr Ala Asp Ser Val Lys 50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65
70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
Cys Ala 85 90 95 Lys Lys Lys Arg Gly Glu Asn Tyr Phe Phe Asp Tyr
Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser 115 104 357 DNA
Homo sapiens 104 gaggtgcagc tgttggagtc tgggggaggc ttggtacagc
ctggggggtc cctgcgtctc 60 tcctgtgcag cctccggatt cacctttcgg
aagtattaga tggggtgggt ccgccaggct 120 ccagggaagg gtctagagtg
ggtctcacag attggtgcga agggtcagtc tacagattac 180 gcagactccg
tgaagggccg gttcaccatc tcccgcgaca attccaagaa cacgctgtat 240
ctgcaaatga acagcctgcg tgccgaggac accgcggtat attactgtgc gaaaaagaag
300 aggggggaga attatttttt tgactactgg ggccagggaa ccctggtcac cgtctcg
357 105 357 DNA Homo sapiens 105 cgagacggtg accagggttc cctggcccca
gtagtcaaaa aaataattct cccccctctt 60 ctttttcgca cagtaatata
ccgcggtgtc ctcggcacgc aggctgttca tttgcagata 120 cagcgtgttc
ttggaattgt cgcgggagat ggtgaaccgg cccttcacgg agtctgcgta 180
atctgtagac tgacccttcg caccaatctg tgagacccac tctagaccct tccctggagc
240 ctggcggacc caccccatct aatacttccg aaaggtgaat ccggaggctg
cacaggagag 300 acgcagggac cccccaggct gtaccaagcc tcccccagac
tccaacagct gcacctc 357 106 119 PRT Homo sapiens 106 Glu Val Gln Leu
Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Arg Tyr 20 25 30
Ser Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Ser Asp Ile Ser Arg Ser Gly Arg Tyr Thr His Tyr Ala Asp Ser
Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Arg Ile Asp Ser Ser Gln Asn
Gly Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser
115 107 357 DNA Homo sapiens 107 gaggtgcagc tgttggagtc tgggggaggc
ttggtacagc ctggggggtc cctgcgtctc 60 tcctgtgcag cctccggatt
cacctttcgg cggtatagta tgtcgtgggt ccgccaggct 120 ccagggaagg
gtctagagtg ggtctcagat atttctcgtt ctggtcggta tacacattac 180
gcagactccg tgaagggccg gttcaccatc tcccgcgaca attccaagaa cacgctgtat
240 ctgcaaatga acagcctgcg tgccgaggac accgcggtat attactgtgc
gaaacgtatt 300 gattcttctc agaatgggtt tgactactgg ggccagggaa
ccctggtcac cgtctcg 357 108 357 DNA Homo sapiens 108 cgagacggtg
accagggttc cctggcccca gtagtcaaac ccattctgag aagaatcaat 60
acgtttcgca cagtaatata ccgcggtgtc ctcggcacgc aggctgttca tttgcagata
120 cagcgtgttc ttggaattgt cgcgggagat ggtgaaccgg cccttcacgg
agtctgcgta 180 atgtgtatac cgaccagaac gagaaatatc tgagacccac
tctagaccct tccctggagc 240 ctggcggacc cacgacatac tataccgccg
aaaggtgaat ccggaggctg cacaggagag 300 acgcagggac cccccaggct
gtaccaagcc tcccccagac tccaacagct gcacctc 357 109 114 PRT Homo
sapiens 109 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Gly Tyr Lys 20 25 30 Met Phe Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu Glu Trp Val Ser 35 40 45 Ala Ile Ser Gly Ser Gly Gly Ser
Thr Tyr Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Lys Gln
Lys Glu Asn Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110
Val Ser 110 345 DNA Homo sapiens 110 gaggtgcagc tgttggagtc
tgggggaggc ttggtacagc ctggggggtc cctgcgtctc 60 tcctgtgcag
cctccggatt caccttttag gggtataaga tgttttgggt ccgccaggct 120
ccagggaagg gtctagagtg ggtctcagct attagtggta gtggtggtag cacatactac
180 gcagactccg tgaagggccg gttcaccatc tcccgcgaca attccaagaa
cacgctgtat 240 ctgcaaatga acagcctgcg tgccgaggac accgcggtat
attactgtgc gaaacagaag 300 gagaattttg actactgggg ccagggaacc
ctggtcaccg tctcg 345 111 345 DNA Homo sapiens 111 cgagacggtg
accagggttc cctggcccca gtagtcaaaa ttctccttct gtttcgcaca 60
gtaatatacc gcggtgtcct cggcacgcag gctgttcatt tgcagataca gcgtgttctt
120 ggaattgtcg cgggagatgg tgaaccggcc cttcacggag tctgcgtagt
atgtgctacc 180 accactacca ctaatagctg agacccactc tagacccttc
cctggagcct ggcggaccca 240 aaacatctta tacccctaaa aggtgaatcc
ggaggctgca caggagagac gcagggaccc 300 cccaggctgt accaagcctc
ccccagactc caacagctgc acctc 345 112 119 PRT Homo sapiens 112 Glu
Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Gly Asp Tyr
20 25 30 Ala Met Trp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45 Ser Val Ile Ser Ser Asn Gly Gly Ser Thr Phe Tyr
Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Arg Val Arg Lys
Arg Thr Pro Glu Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val
Thr Val Ser 115 113 357 DNA Homo sapiens 113 gaggtgcagc tgttggagtc
tgggggaggc ttggtacagc ctggggggtc cctgcgtctc 60 tcctgtgcag
cctccggatt
cacctttggg gattatgcta tgtggtgggt ccgccaggct 120 ccagggaagg
gtctagagtg ggtctcagtg attagttcga atggtgggag tacattttac 180
gcagactccg tgaagggccg gttcaccatc tcccgcgaca attccaagaa cacgctgtat
240 ctgcaaatga acagcctgcg tgccgaggac accgcggtat attactgtgc
gaaacgtgtt 300 cgtaagagga ctcctgagtt tgactactgg ggccagggaa
ccctggtcac cgtctcg 357 114 357 DNA Homo sapiens 114 cgagacggtg
accagggttc cctggcccca gtagtcaaac tcaggagtcc tcttacgaac 60
acgtttcgca cagtaatata ccgcggtgtc ctcggcacgc aggctgttca tttgcagata
120 cagcgtgttc ttggaattgt cgcgggagat ggtgaaccgg cccttcacgg
agtctgcgta 180 aaatgtactc ccaccattcg aactaatcac tgagacccac
tctagaccct tccctggagc 240 ctggcggacc caccacatag cataatcccc
aaaggtgaat ccggaggctg cacaggagag 300 acgcagggac cccccaggct
gtaccaagcc tcccccagac tccaacagct gcacctc 357 115 119 PRT Homo
sapiens 115 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Arg Arg Tyr 20 25 30 Lys Met Gly Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Gly Arg Asn Gly Thr
Lys Thr Asn Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys
Ile Tyr Thr Gly Lys Pro Ala Ala Phe Asp Tyr Trp Gly Gln 100 105 110
Gly Thr Leu Val Thr Val Ser 115 116 357 DNA Homo sapiens 116
gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgcgtctc
60 tcctgtgcag cctccggatt cacctttagg aggtataaga tgggttgggt
ccgccaggct 120 ccagggaagg gtctagagtg ggtctcagcg attgggagga
atggtacgaa gacaaattac 180 gcagactccg tgaagggccg gttcaccatc
tcccgcgaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgcg
tgccgaggac accgcggtat attactgtgc gaaaatttat 300 acggggaagc
ctgctgcgtt tgactactgg ggccagggaa ccctggtcac cgtctcg 357 117 357 DNA
Homo sapiens 117 cgagacggtg accagggttc cctggcccca gtagtcaaac
gcagcaggct tccccgtata 60 aattttcgca cagtaatata ccgcggtgtc
ctcggcacgc aggctgttca tttgcagata 120 cagcgtgttc ttggaattgt
cgcgggagat ggtgaaccgg cccttcacgg agtctgcgta 180 atttgtcttc
gtaccattcc tcccaatcgc tgagacccac tctagaccct tccctggagc 240
ctggcggacc caacccatct tatacctcct aaaggtgaat ccggaggctg cacaggagag
300 acgcagggac cccccaggct gtaccaagcc tcccccagac tccaacagct gcacctc
357 118 118 PRT Homo sapiens 118 Glu Val Gln Leu Leu Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Lys Lys Tyr 20 25 30 Met Ser Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser 35 40 45 Ala Ile
Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys 50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65
70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
Cys Ala 85 90 95 Lys Met Leu Arg Thr Lys Asn Lys Val Phe Asp Tyr
Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser 115 119 357 DNA
Homo sapiens 119 gaggtgcagc tgttggagtc tgggggaggc ttggtacagc
ctggggggtc cctgcgtctc 60 tcctgtgcag cctccggatt cacctttaag
aagtattaga tgtcttgggt ccgccaggct 120 ccagggaagg gtctagagtg
ggtctcagct attagtggta gtggtggtag cacatactac 180 gcagactccg
tgaagggccg gttcaccatc tcccgcgaca attccaagaa cacgctgtat 240
ctgcaaatga acagcctgcg tgccgaggac accgcggtat attactgtgc gaaaatgctg
300 aggactaaga ataaggtgtt tgactactgg ggccagggaa ccctggtcac cgtctcg
357 120 357 DNA Homo sapiens 120 cgagacggtg accagggttc cctggcccca
gtagtcaaac accttattct tagtcctcag 60 cattttcgca cagtaatata
ccgcggtgtc ctcggcacgc aggctgttca tttgcagata 120 cagcgtgttc
ttggaattgt cgcgggagat ggtgaaccgg cccttcacgg agtctgcgta 180
gtatgtgcta ccaccactac cactaatagc tgagacccac tctagaccct tccctggagc
240 ctggcggacc caagacatct aatacttctt aaaggtgaat ccggaggctg
cacaggagag 300 acgcagggac cccccaggct gtaccaagcc tcccccagac
tccaacagct gcacctc 357 121 119 PRT Homo sapiens 121 Glu Val Gln Leu
Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Arg Tyr 20 25 30
Lys Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Ser Ala Ile Gly Arg Asn Gly Thr Lys Thr Asn Tyr Ala Asp Ser
Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ile Tyr Thr Gly Lys Pro Ala
Ala Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser
115 122 357 DNA Homo sapiens 122 gaggtgcagc tgttggagtc tgggggaggc
ttggtacagc ctggggggtc cctgcgtctc 60 tcctgtgcag cctccggatt
cacctttagg aggtataaga tgggttgggt ccgccaggct 120 ccagggaagg
gtctagagtg ggtctcagcg attgggagga atggtacgaa gacaaattac 180
gcagactccg tgaagggccg gttcaccatc tcccgcgaca attccaagaa cacgctgtat
240 ctgcaaatga acagcctgcg tgccgaggac accgcggtat attactgtgc
gaaaatttat 300 acggggaagc ctgctgcgtt tgactactgg ggccagggaa
ccctggtcac cgtctcg 357 123 357 DNA Homo sapiens 123 cgagacggtg
accagggttc cctggcccca gtagtcaaac gcagcaggct tccccgtata 60
aattttcgca cagtaatata ccgcggtgtc ctcggcacgc aggctgttca tttgcagata
120 cagcgtgttc ttggaattgt cgcgggagat ggtgaaccgg cccttcacgg
agtctgcgta 180 atttgtcttc gtaccattcc tcccaatcgc tgagacccac
tctagaccct tccctggagc 240 ctggcggacc caacccatct tatacctcct
aaaggtgaat ccggaggctg cacaggagag 300 acgcagggac cccccaggct
gtaccaagcc tcccccagac tccaacagct gcacctc 357 124 118 PRT Homo
sapiens 124 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Tyr Arg 20 25 30 Met Gly Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu Glu Trp Val Ser 35 40 45 Ser Ile Ser Ser Arg Gly Arg His
Thr Ser Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Lys Arg
Val Pro Gly Arg Gly Arg Ser Phe Asp Tyr Trp Gly Gln Gly 100 105 110
Thr Leu Val Thr Val Ser 115 125 357 DNA Homo sapiens 125 gaggtgcagc
tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgcgtctc 60
tcctgtgcag cctccggatt caccttttag agttatcgga tgggttgggt ccgccaggct
120 ccagggaagg gtctagagtg ggtctcaagt atttcgtcga ggggtaggca
tacatcttac 180 gcagactccg tgaagggccg gttcaccatc tcccgcgaca
attccaagaa cacgctgtat 240 ctgcaaatga acagcctgcg tgccgaggac
accgcggtat attactgtgc gaaaagggtt 300 ccgggtcggg ggcgttcttt
tgactactgg ggccagggaa ccctggtcac cgtctcg 357 126 357 DNA Homo
sapiens 126 cgagacggtg accagggttc cctggcccca gtagtcaaaa gaacgccccc
gacccggaac 60 ccttttcgca cagtaatata ccgcggtgtc ctcggcacgc
aggctgttca tttgcagata 120 cagcgtgttc ttggaattgt cgcgggagat
ggtgaaccgg cccttcacgg agtctgcgta 180 agatgtatgc ctacccctcg
acgaaatact tgagacccac tctagaccct tccctggagc 240 ctggcggacc
caacccatcc gataactcta aaaggtgaat ccggaggctg cacaggagag 300
acgcagggac cccccaggct gtaccaagcc tcccccagac tccaacagct gcacctc 357
127 119 PRT Homo sapiens 127 Glu Val Gln Leu Leu Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Pro Phe Arg Arg Tyr 20 25 30 Arg Met Arg Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile
Ser Pro Gly Gly Lys His Thr Thr Tyr Ala Asp Ser Val 50 55 60 Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70
75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95 Ala Lys Gly Glu Gly Gly Ala Ser Ser Ala Phe Asp Tyr
Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser 115 128 357 DNA
Homo sapiens 128 gaggtgcagc tgttggagtc tgggggaggc ttggtacagc
ctggggggtc cctgcgtctc 60 tcctgtgcag cctccggatt cccctttcgt
cggtatcgga tgaggtgggt ccgccaggct 120 ccagggaagg gtctagagtg
ggtctcaggt atttctccgg gtggtaagca tacaacgtac 180 gcagactccg
tgaagggccg gttcaccatc tcccgcgaca attccaagaa cacgctgtat 240
ctgcaaatga acagcctgcg tgccgaggac accgcggtat attactgtgc gaaaggtgag
300 gggggggcga gttctgcgtt tgactactgg ggccagggaa ccctggtcac cgtctcg
357 129 357 DNA Homo sapiens 129 cgagacggtg accagggttc cctggcccca
gtagtcaaac gcagaactcg cccccccctc 60 acctttcgca cagtaatata
ccgcggtgtc ctcggcacgc aggctgttca tttgcagata 120 cagcgtgttc
ttggaattgt cgcgggagat ggtgaaccgg cccttcacgg agtctgcgta 180
cgttgtatgc ttaccacccg gagaaatacc tgagacccac tctagaccct tccctggagc
240 ctggcggacc cacctcatcc gataccgacg aaaggggaat ccggaggctg
cacaggagag 300 acgcagggac cccccaggct gtaccaagcc tcccccagac
tccaacagct gcacctc 357 130 118 PRT Homo sapiens 130 Glu Val Gln Leu
Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Tyr Gly 20 25 30
Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser 35
40 45 Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val
Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys Ala 85 90 95 Lys Arg His Ser Ser Glu Ala Arg Gln
Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser 115
131 357 DNA Homo sapiens 131 gaggtgcagc tgttggagtc tgggggaggc
ttggtacagc ctggggggtc cctgcgtctc 60 tcctgtgcag cctccggatt
caccttttag cggtatggga tggtttgggt ccgccaggct 120 ccagggaagg
gtctagagtg ggtctcagct attagtggta gtggtggtag cacatactac 180
gcagactccg tgaagggccg gttcaccatc tcccgcgaca attccaagaa cacgctgtat
240 ctgcaaatga acagcctgcg tgccgaggac accgcggtat attactgtgc
gaaacggcat 300 agttctgagg ctaggcagtt tgactactgg ggccagggaa
ccctggtcac cgtctcg 357 132 357 DNA Homo sapiens 132 cgagacggtg
accagggttc cctggcccca gtagtcaaac tgcctagcct cagaactatg 60
ccgtttcgca cagtaatata ccgcggtgtc ctcggcacgc aggctgttca tttgcagata
120 cagcgtgttc ttggaattgt cgcgggagat ggtgaaccgg cccttcacgg
agtctgcgta 180 gtatgtgcta ccaccactac cactaatagc tgagacccac
tctagaccct tccctggagc 240 ctggcggacc caaaccatcc cataccgcta
aaaggtgaat ccggaggctg cacaggagag 300 acgcagggac cccccaggct
gtaccaagcc tcccccagac tccaacagct gcacctc 357 133 5 PRT Artificial
Sequence CDR1 VARIANT 1, 2, 3, 5 Xaa = Any Amino Acid 133 Xaa Xaa
Xaa Leu Xaa 1 5 134 7 PRT Artificial Sequence CDR2 VARIANT 1, 4 Xaa
= Any Amino Acid 134 Xaa Ala Ser Xaa Leu Gln Ser 1 5 135 9 PRT
Artificial Sequence CDR3 VARIANT 3, 4, 5, 6, 8 Xaa = Any Amino Acid
135 Gln Gln Xaa Xaa Xaa Xaa Pro Xaa Thr 1 5 136 5 PRT Artificial
Sequence CDR1 136 Ser Ser Tyr Leu Asn 1 5 137 7 PRT Artificial
Sequence CDR2 137 Arg Ala Ser Pro Leu Gln Ser 1 5 138 9 PRT
Artificial Sequence CDR3 138 Gln Gln Thr Tyr Ser Val Pro Pro Thr 1
5 139 5 PRT Artificial Sequence CDR1 139 Ser Ser Tyr Leu Asn 1 5
140 7 PRT Artificial Sequence CDR2 140 Arg Ala Ser Pro Leu Gln Ser
1 5 141 9 PRT Artificial Sequence CDR3 141 Gln Gln Thr Tyr Arg Ile
Pro Pro Thr 1 5 142 5 PRT Artificial Sequence CDR1 142 Phe Lys Ser
Leu Lys 1 5 143 7 PRT Artificial Sequence CDR2 143 Asn Ala Ser Tyr
Leu Gln Ser 1 5 144 9 PRT Artificial Sequence CDR3 144 Gln Gln Val
Val Tyr Trp Pro Val Thr 1 5 145 5 PRT Artificial Sequence CDR1 145
Tyr Tyr His Leu Lys 1 5 146 7 PRT Artificial Sequence CDR2 146 Lys
Ala Ser Thr Leu Gln Ser 1 5 147 9 PRT Artificial Sequence CDR3 147
Gln Gln Val Arg Lys Val Pro Arg Thr 1 5 148 5 PRT Artificial
Sequence CDR1 148 Arg Arg Tyr Leu Lys 1 5 149 7 PRT Artificial
Sequence CDR2 149 Gln Ala Ser Val Leu Gln Ser 1 5 150 9 PRT
Artificial Sequence CDR3 150 Gln Gln Gly Leu Tyr Pro Pro Ile Thr 1
5 151 5 PRT Artificial Sequence CDR1 151 Tyr Asn Trp Leu Lys 1 5
152 7 PRT Artificial Sequence CDR2 152 Arg Ala Ser Ser Leu Gln Ser
1 5 153 9 PRT Artificial Sequence CDR3 153 Gln Gln Asn Val Val Ile
Pro Arg Thr 1 5 154 5 PRT Artificial Sequence CDR1 154 Leu Trp His
Leu Arg 1 5 155 7 PRT Artificial Sequence CDR2 155 His Ala Ser Leu
Leu Gln Ser 1 5 156 9 PRT Artificial Sequence CDR3 156 Gln Gln Ser
Ala Val Tyr Pro Lys Thr 1 5 157 5 PRT Artificial Sequence CDR1 157
Phe Arg Tyr Leu Ala 1 5 158 7 PRT Artificial Sequence CDR2 158 His
Ala Ser His Leu Gln Ser 1 5 159 9 PRT Artificial Sequence CDR3 159
Gln Gln Arg Leu Leu Tyr Pro Lys Thr 1 5 160 5 PRT Artificial
Sequence CDR1 160 Phe Tyr His Leu Ala 1 5 161 7 PRT Artificial
Sequence CDR2 161 Pro Ala Ser Lys Leu Gln Ser 1 5 162 9 PRT
Artificial Sequence CDR3 162 Gln Gln Arg Ala Arg Trp Pro Arg Thr 1
5 163 5 PRT Artificial Sequence CDR1 163 Ile Trp His Leu Asn 1 5
164 7 PRT Artificial Sequence CDR2 164 Arg Ala Ser Arg Leu Gln Ser
1 5 165 9 PRT Artificial Sequence CDR3 165 Gln Gln Val Ala Arg Val
Pro Arg Thr 1 5 166 5 PRT Artificial Sequence CDR1 166 Tyr Arg Tyr
Leu Arg 1 5 167 7 PRT Artificial Sequence CDR2 167 Lys Ala Ser Ser
Leu Gln Ser 1 5 168 9 PRT Artificial Sequence CDR3 168 Gln Gln Tyr
Val Gly Tyr Pro Arg Thr 1 5 169 5 PRT Artificial Sequence CDR1 169
Leu Lys Tyr Leu Lys 1 5 170 7 PRT Artificial Sequence CDR2 170 Asn
Ala Ser His Leu Gln Ser 1 5 171 9 PRT Artificial Sequence CDR3 171
Gln Gln Thr Thr Tyr Tyr Pro Ile Thr 1 5 172 5 PRT Artificial
Sequence CDR1 172 Leu Arg Tyr Leu Arg 1 5 173 7 PRT Artificial
Sequence CDR2 173 Lys Ala Ser Trp Leu Gln Ser 1 5 174 9 PRT
Artificial Sequence CDR3 174 Gln Gln Val Leu Tyr Tyr Pro Gln Thr 1
5 175 5 PRT Artificial Sequence CDR1 175 Leu Arg Ser Leu Lys 1 5
176 7 PRT Artificial Sequence CDR2 176 Ala Ala Ser Arg Leu Gln Ser
1 5 177 9 PRT Artificial Sequence CDR3 177 Gln Gln Val Val Tyr Trp
Pro Ala Thr 1 5 178 5 PRT Artificial Sequence CDR1 178 Phe Arg His
Leu Lys 1 5 179 7 PRT Artificial Sequence CDR2 179 Ala Ala Ser Arg
Leu Gln Ser 1 5 180 9 PRT Artificial Sequence CDR3 180 Gln Gln Val
Ala Leu Tyr Pro Lys Thr 1 5 181 5 PRT Artificial Sequence CDR1 181
Arg Lys Tyr Leu Arg 1 5 182 7 PRT Artificial Sequence CDR2 182 Thr
Ala Ser Ser Leu Gln Ser 1 5 183 9 PRT Artificial Sequence CDR3 183
Gln Gln Asn Leu Phe Trp Pro Arg Thr 1 5 184 5 PRT Artificial
Sequence CDR1 184 Arg Arg Tyr Leu Asn 1 5 185 7 PRT Artificial
Sequence CDR2 185 Ala Ala Ser Ser Leu Gln Ser 1 5 186 9 PRT
Artificial Sequence CDR3 186 Gln Gln Met Leu Phe Tyr Pro Lys Thr 1
5 187 5 PRT Artificial Sequence CDR1 187 Ile Lys His Leu Lys 1 5
188 7 PRT Artificial Sequence CDR2 188 Gly Ala Ser Arg Leu Gln Ser
1 5 189 9 PRT Artificial Sequence CDR3 189 Gln Gln Gly Ala Arg Trp
Pro Gln Thr 1 5 190 5 PRT Artificial Sequence CDR1 190 Tyr Tyr His
Leu Lys 1 5 191 7 PRT Artificial Sequence CDR2 191 Lys Ala Ser Thr
Leu Gln Ser 1 5 192 9 PRT Artificial Sequence CDR3 192 Gln Gln Val
Arg Lys Val Pro Arg Thr 1 5 193 5 PRT Artificial Sequence CDR1 193
Tyr Lys His Leu Lys 1 5 194 7 PRT Artificial Sequence CDR2 194 Asn
Ala Ser His Leu Gln Ser 1 5 195 9 PRT Artificial Sequence CDR3 195
Gln Gln Val Gly Arg Tyr Pro Lys Thr 1 5 196 5 PRT Artificial
Sequence CDR1 196 Phe Lys Ser Leu Lys 1 5 197 7 PRT Artificial
Sequence CDR2 197 Asn Ala Ser Tyr Leu Gln Ser 1 5 198 9 PRT
Artificial Sequence CDR3 198 Gln Gln Val Val Tyr Trp Pro Val Thr 1
5 199 6 PRT Artificial Sequence CDR1 VARIANT 1, 2, 4, 5, 6 Xaa =
Any Amino Acid 199 Xaa Xaa Tyr Xaa Xaa Xaa 1 5 200 17 PRT
Artificial Sequence CDR2 VARIANT 1, 3, 4, 5, 7, 8, 10 Xaa = Any
Amino Acid 200 Xaa Ile Xaa Xaa Xaa Gly Xaa Xaa Thr Xaa Tyr Ala Asp
Ser Val Lys 1 5 10 15 Gly 201 11 PRT Artificial Sequence CDR3
VARIANT 1, 2, 3, 4, 5, 6, 7, 8 Xaa = Any Amino Acid 201 Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Phe
Asp Tyr 1 5 10 202 6 PRT Artificial Sequence CDR1 202 Trp Val Tyr
Gln Met Asp 1 5 203 17 PRT Artificial Sequence CDR2 203 Ser Ile Ser
Ala Phe Gly Ala Lys Thr Leu Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly
204 7 PRT Artificial Sequence CDR3 204 Leu Ser Gly Lys Phe Asp Tyr
1 5 205 6 PRT Artificial Sequence CDR1 205 Trp Ser Tyr Gln Met Thr
1 5 206 17 PRT Artificial Sequence CDR2 206 Ser Ile Ser Ser Phe Gly
Ser Ser Thr Leu Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly 207 11 PRT
Artificial Sequence CDR3 207 Gly Arg Asp His Asn Tyr Ser Leu Phe
Asp Tyr 1 5 10 208 22 DNA Artificial Sequence VK-DLIBF primer 208
cggccatggc gtcaacggac at 22 209 23 DNA Artificial Sequence VKXho1R
primer 209 atgtgcgctc gagcgtttga ttt 23 210 15 PRT Artificial
Sequence Modified 1gGC1 hinge 210 Glu Pro Lys Ser Gly Asp Lys Thr
His Thr Cys Pro Pro Cys Pro 1 5 10 15 211 108 PRT Homo sapiens 211
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp 1 5
10 15 Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Asp Ser Tyr
Leu 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile Tyr 35 40 45 Ser Ala Ser Glu Leu Gln Ser Gly Val Pro Ser
Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro Glu 65 70 75 80 Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln Val Val Trp Arg Pro Phe Thr 85 90 95 Phe Gly Gln Gly Thr
Lys Val Glu Ile Lys Arg Cys 100 105 212 357 DNA Homo sapiens 212
tggagcgcgt cgacggacat ccagatgacc cagtctccat cctctctgtc tgcatctgta
60 ggagaccgtg tcaccatcac ttgccgggca agtcagagca ttgatagtta
tttacattgg 120 taccagcaga aaccagggaa agcccctaag ctcctgatct
atagtgcatc cgagttgcaa 180 agtggggtcc catcacgttt cagtggcagt
ggatctggga cagatttcac tctcaccatc 240 agcagtctgc aacctgaaga
ttttgctacg tactactgtc aacaggttgt gtggcgtcct 300 tttacgttcg
gccaagggac caaggtggaa atcaaacggt gctaataagg atccggc 357 213 357 DNA
Homo sapiens 213 gccggatcct tattagcacc gtttgatttc caccttggtc
ccttggccga acgtaaaagg 60 acgccacaca acctgttgac agtagtacgt
agcaaaatct tcaggttgca gactgctgat 120 ggtgagagtg aaatctgtcc
cagatccact gccactgaaa cgtgatggga ccccactttg 180 caactcggat
gcactataga tcaggagctt aggggctttc cctggtttct gctggtacca 240
atgtaaataa ctatcaatgc tctgacttgc ccggcaagtg atggtgacac ggtctcctac
300 agatgcagac agagaggatg gagactgggt catctggatg tccgtcgacg cgctcca
357 214 39 DNA Artificial Sequence Primer 214 tggagcgcgt cgacggacat
ccagatgacc cagtctcca 39 215 39 DNA Artificial Sequence Primer 215
ttagcagccg gatccttatt agcaccgttt gatttccac 39 216 27 DNA Artificial
Sequence HA tag 216 tatccttatg atgttcctga ttatgca 27 217 9 PRT
Artificial Sequence HA tag 217 Tyr Pro Tyr Asp Val Pro Asp Tyr Ala
1 5 218 120 PRT Homo sapiens 218 Glu Val Gln Leu Leu Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Glu Trp Tyr 20 25 30 Trp Met Gly Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala
Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65
70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Lys Val Lys Leu Gly Gly Gly Pro Asn Phe Asp
Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115
120
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